Xeroderma pigmentosum: a case report and review of the literature.

PubMed

Feller, L; Wood, N H; Motswaledi, M H; Khammissa, R A G; Meyer, M; Lemmer, J

2010-06-01

Inherited molecular defects in nucleotide excision repair genes cause the autosomal recessive condition xeroderma pigmentosum. Xeroderma pigmentosum is characterized by photo-hypersensitivity of sun-exposed tissues, and by a several thousand-fold increase in the risk of developing malignant neoplasms of the skin and of the eyes. Mutations in xeroderma pigmentosum genes that regulate nucleotide excision repair, not only predispose persons with xeroderma pigmentosum to multiple malignancies, but also promote premature cutaneous and ocular ageing, and in some cases promote progressive neurodegenerative changes. This paper describes a case of xeroderma pigmentosum with advanced cutaneous squamous cell carcinoma, actinic cheilitis and ocular lesions in a 19-year-old black woman. The extensive ultraviolet radiation-induced skin and eye damage are evidence of neglect of sun-protection and lack of appropriate medical care from childhood.

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene

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

Lommel, L.; Hanawalt, P.C.

1993-02-01

People that suffer from xeroderma pigmentosum (XP) are sun sensitive and experience elevated incidences of cancer, particularly skin cancers on sun-light exposed parts of their bodies. Cultured cells from XP patients are found to be subtantially more sensitive to lethal and mutagenic effects of ultraviolet (UV) radiation than are cells from unaffected individuals. Using the cells from XP individuals, researchers study the roles that cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts play in UV resistance. The results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs form the transcribed strands of expressedmore » genes is essential for UV resistance. 36 refs., 5 figs., 1 tab.« less

Functional characterization of polymorphisms in DNA repair genes using cytogenetic challenge assays.

PubMed

Au, William W; Salama, Salama A; Sierra-Torres, Carlos H

2003-11-01

A major barrier to understanding the role of polymorphic DNA repair genes for environmental cancer is that the functions of variant genotypes are largely unknown. Using our cytogenetic challenge assays, we conducted an investigation to address the deficiency. Using X-rays or ultraviolet (UV) light, we irradiated blood lymphocytes from 80 nonsmoking donors to challenge the cells to repair the induced DNA damage, and we analyzed expression of chromosome aberrations (CA) specific to the inducing agents. We have genotyped polymorphic DNA repair genes preferentially involved with base excision repair (BER) and nucleotide excision repair (NER) activities (XRCC1, XRCC3, APE1, XPD) corresponding to the repair of X-ray- and UV light-induced DNA damage, respectively. We expected that defects in specific DNA repair pathways due to polymorphisms would cause corresponding increases of specific CA. From our data, XRCC1 399Gln and XRCC3 241Met were associated with significant increases in chromosome deletions compared with the corresponding homozygous wild types (18.27 1.1 vs 14.79 1.2 and 18.22 0.99 vs 14.20 1.39, respectively); XPD 312Asn and XPD 751Gln were associated with significant increases in chromatid breaks compared with wild types (16.09 1.36 vs 11.41 0.98 and 16.87 1.27 vs 10.54 0.87, respectively), p < 0.05. The data indicate that XRCC1 399Gln and XRCC3 241Met are significantly defective in BER, and the XPD 312Asn and XPD 751Gln are significantly defective in NER. In addition, the variant genotypes interact significantly, with limited overlap of the two different repair pathways.

Ultraviolet mutagenesis studies of [psi], a cytoplasmic determinant of Saccharomyces cerevisiae.

PubMed

Tuite, M F; Cox, B S

1980-07-01

UV mutagenesis was used to probe the molecular nature of [psi], a nonmitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The UV-induced mutation from [psi+] to [psi-] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi-] mutation was demonstrated by photoreactivation. UV-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. UV-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA.

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

PubMed

Dupuy, Aurélie; Sarasin, Alain

2015-06-01

Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system.

PubMed

Guthrie, O'neil W

2017-10-01

In response to DNA damage from ultraviolet (UV) radiation, bacteria deploy the SOS response in order to limit cell death. This bacterial SOS response is characterized by an increase in the recA gene that transactivates expression of multiple DNA repair genes. The current series of experiments demonstrate that a mammalian organ system (the cochlea) that is not evolutionarily conditioned to UV radiation can elicit SOS responses that are reminiscent of that of bacteria. This mammalian SOS response is characterized by an increase in the p53 gene with activation of multiple DNA repair genes that harbor p53 response elements in their promoters. Furthermore, the experimental results provide support for the notion of a convergent trigger paradox, where independent SOS triggers facilitate disparate physiologic sequelae (loss vs. recovery of function). Therefore, it is proposed that the mammalian SOS response is multifunctional and manipulation of this endogenous response could be exploited in future biomedical interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats.

PubMed

Palmer, Jonathan M; Drees, Kevin P; Foster, Jeffrey T; Lindner, Daniel L

2018-01-02

Bat white-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, has decimated North American hibernating bats since its emergence in 2006. Here, we utilize comparative genomics to examine the evolutionary history of this pathogen in comparison to six closely related nonpathogenic species. P. destructans displays a large reduction in carbohydrate-utilizing enzymes (CAZymes) and in the predicted secretome (~50%), and an increase in lineage-specific genes. The pathogen has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which is known to contribute to repair of DNA lesions induced by ultraviolet (UV) light. Consistent with a nonfunctional AER pathway, P. destructans is extremely sensitive to UV light, as well as the DNA alkylating agent methyl methanesulfonate (MMS). The differential susceptibility of P. destructans to UV light in comparison to other hibernacula-inhabiting fungi represents a potential "Achilles' heel" of P. destructans that might be exploited for treatment of bats with WNS.

Xeroderma pigmentosum, complementation group D expression in H1299 lung cancer cells following benzo[a]pyrene exposure as well as in head and neck cancer patients.

PubMed

Lin, Chang-Shen; Chiou, Wen-Yen; Lee, Ka-Wo; Chen, Tzu-Fen; Lin, Yuan-Jen; Huang, Jau-Ling

2016-01-01

DNA repair genes play critical roles in response to carcinogen-induced and anticancer therapy-induced DNA damage. Benzo[a]pyrene (BaP), the most carcinogenic polycyclic aromatic hydrocarbon (PAH), is classified as a group 1 carcinogen by International Agency for Research on Cancer. The aims of this study were to (1) evaluate the effects of BaP on DNA repair activity and expression of DNA repair genes in vitro and (2) examine the role of xeroderma pigmentosum, complementation group D (XPD) mRNA expression in human head and neck cancers. Host cell reactivation assay showed that BaP inhibited nucleotide excision repair in H1299 lung cancer cells. DNA repair through the non-homologous end-joining pathway was not affected by BaP. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot demonstrated that XPD was downregulated by BaP treatment. BaP exposure did not apparently affect expression of another 11 DNA repair genes. BaP treatment increased the DNA damage marker γ-H2AX and ultraviolet (UV) sensitivity, supporting an impairment of DNA repair in BaP-treated cells. XPD expression was also examined by quantitative RT-PCR in 68 head and neck cancers, and a lower XPD mRNA level was found in smokers' cancer specimens. Importantly, reduced XPD expression was correlated with patient 5-year overall survival rate (35 vs. 56%) and was an independent prognostic factor (hazard ratio: 2.27). Data demonstrated that XPD downregulation was correlated with BaP exposure and human head and neck cancer survival.

Divergence in DNA photorepair efficiency among genotypes from contrasting UV radiation environments in nature.

PubMed

Miner, Brooks E; Kulling, Paige M; Beer, Karlyn D; Kerr, Benjamin

2015-12-01

Populations of organisms routinely face abiotic selection pressures, and a central goal of evolutionary biology is to understand the mechanistic underpinnings of adaptive phenotypes. Ultraviolet radiation (UVR) is one of earth's most pervasive environmental stressors, potentially damaging DNA in any organism exposed to solar radiation. We explored mechanisms underlying differential survival following UVR exposure in genotypes of the water flea Daphnia melanica derived from natural ponds of differing UVR intensity. The UVR tolerance of a D. melanica genotype from a high-UVR habitat depended on the presence of visible and UV-A light wavelengths necessary for photoenzymatic repair of DNA damage, a repair pathway widely shared across the tree of life. We then measured the acquisition and repair of cyclobutane pyrimidine dimers, the primary form of UVR-caused DNA damage, in D. melanica DNA following experimental UVR exposure. We demonstrate that genotypes from high-UVR habitats repair DNA damage faster than genotypes from low-UVR habitats in the presence of visible and UV-A radiation necessary for photoenzymatic repair, but not in dark treatments. Because differences in repair rate only occurred in the presence of visible and UV-A radiation, we conclude that differing rates of DNA repair, and therefore differential UVR tolerance, are a consequence of variation in photoenzymatic repair efficiency. We then rule out a simple gene expression hypothesis for the molecular basis of differing repair efficiency, as expression of the CPD photolyase gene photorepair did not differ among D. melanica lineages, in both the presence and absence of UVR. © 2015 John Wiley & Sons Ltd.

Toll-Like Receptor-4 deficiency enhances repair of ultraviolet radiation induced cutaneous DNA damage by nucleotide excision repair mechanism

PubMed Central

Ahmad, Israr; Simanyi, Eva; Guroji, Purushotham; Tamimi, Iman A; delaRosa, Hillary J; Nagar, Anusuiya; Nagar, Priyamvada; Katiyar, Santosh K; Elmets, Craig A; Yusuf, Nabiha

2014-01-01

UVB-induced DNA damage plays a critical role in development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4-/-) and TLR4 competent (TLR4+/+) mice were subjected to 90 mJ/cm2 UVB radiation locally, DNA damage in the form of CPD, were repaired more efficiently in the skin and bone marrow dendritic cells (BMDC) of TLR4-/- mice in comparison to TLR4+/+ mice. Expression of DNA repair gene XPA (Xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDC of TLR4+/+ mice than TLR4-/- mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDC from UV-irradiated TLR4-/- mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (p<0.05) than BMDC from TLR4+/+ mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDC of TLR4-/- mice (before UVB exposure) inhibited repair of CPD, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4+/+ BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer. PMID:24326454

Ancestor of land plants acquired the DNA-3-methyladenine glycosylase (MAG) gene from bacteria through horizontal gene transfer.

PubMed

Fang, Huimin; Huangfu, Liexiang; Chen, Rujia; Li, Pengcheng; Xu, Shuhui; Zhang, Enying; Cao, Wei; Liu, Li; Yao, Youli; Liang, Guohua; Xu, Chenwu; Zhou, Yong; Yang, Zefeng

2017-08-24

The origin and evolution of land plants was an important event in the history of life and initiated the establishment of modern terrestrial ecosystems. From water to terrestrial environments, plants needed to overcome the enhanced ultraviolet (UV) radiation and many other DNA-damaging agents. Evolving new genes with the function of DNA repair is critical for the origin and radiation of land plants. In bacteria, the DNA-3-methyladenine glycosylase (MAG) recognizes of a variety of base lesions and initiates the process of the base excision repair for damaged DNA. The homologs of MAG gene are present in all major lineages of streptophytes, and both the phylogenic and sequence similarity analyses revealed that green plant MAG gene originated through an ancient horizontal gene transfer (HGT) event from bacteria. Experimental evidence demonstrated that the expression of the maize ZmMAG gene was induced by UV and zeocin, both of which are known as DNA-damaging agents. Further investigation revealed that Streptophyta MAG genes had undergone positive selection during the initial evolutionary period in the ancestor of land plants. Our findings demonstrated that the ancient HGT of MAG to the ancestor of land plants probably played an important role in preadaptation to DNA-damaging agents in terrestrial environments.

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair.

PubMed Central

Hidema, J.; Kumagai, T.; Sutherland, J. C.; Sutherland, B. M.

1997-01-01

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair. PMID:12223592

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

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

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.

1987-12-01

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C tomore » A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.« less

Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole

PubMed Central

Steffens, Laura S.; Nicholson, Samantha; Paul, Lynthia V.; Nord, Carl Erik; Patrick, Sheila; Abratt, Valerie R.

2010-01-01

Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium. PMID:20435137

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

PubMed

Prakash, L

1974-12-01

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

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

PubMed

Schons-Fonseca, Luciane; da Silva, Josefa B; Milanez, Juliana S; Domingos, Renan H; Smith, Janet L; Nakaya, Helder I; Grossman, Alan D; Ho, Paulo L; da Costa, Renata M A

2016-02-18

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Postreplication Repair of Ultraviolet Damage in Haemophilus influenzae

PubMed Central

Leclerc, J. Eugene; Setlow, Jane K.

1972-01-01

The deoxyribonucleic acid (DNA) synthesized following ultraviolet (UV) irradiation of wild-type (Rd) and recombination-defective strains of Haemophilus influenzae has been analyzed by alkaline sucrose gradient sedimentation. Strain Rd and a UV-resistant, recombination-defective strain Rd(DB117) rec− are able to carry out postreplication repair, i.e., close the single-strand gaps in the newly synthesized DNA; in the UV-sensitive, recombination-defective strain DB117, the gaps remain open. The lack of postreplication repair in this strain may be the result of degradation of the newly synthesized DNA. PMID:4537422

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Alterations in ATR in nasal NK/T-cell lymphoma and chronic active Epstein-Barr virus infection.

PubMed

Liu, Angen; Takakuwa, Tetsuya; Luo, Wen-Juan; Fujita, Shigeki; Aozasa, Katsuyuki

2006-07-01

Nasal natural killer (NK)/T-cell lymphoma (NKTCL) and chronic active Epstein-Barr virus infection (CAEBV) are relatively frequent, especially in Asia, and are poor in prognosis. Both diseases are proliferative diseases of NK/T cells that show highly complicated karyotypes, suggesting the involvement of chromosomal instability. ATR is an important gene for DNA damage response and chromosomal stability. To evaluate the role of ATR gene alterations in the pathogenesis of NKTCL and CAEBV, the whole coding region of the ATR gene was examined in cell lines derived from NKTCL and CAEBV, as well as tumor samples from patients. ATR alterations were detected in two of eight NKTCL and in one of three CAEBV lines. Most aberrant transcripts observed were deletions resulting from aberrant splicing. ATR alterations were also detected in four of 10 NKTCL clinical samples. Both NKTCL and CAEBV cell lines with ATR alterations showed a delay or abrogation in repair of ionizing radiation-induced DNA double-strand breaks and ultraviolet-induced DNA single-strand breaks, and both exhibited a defect in p53 accumulation. These findings show that alterations in the ATR gene result in an abnormal response to DNA double-strand break and single-strand break repair, suggesting a role for ATR gene alterations in NKTCL lymphomagenesis.

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

PubMed Central

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

2014-01-01

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

Regulation and disregulation of mammalian nucleotide excision repair: A pathway to nongermline breast carcinogenesis

DOE PAGES

Latimer, Jean J.; Majekwana, Vongai J.; Pabon-Padin, Yashira R.; ...

2014-12-19

Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NERmore » pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this article, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.« less

Water quality implications of culvert repair options : vinyl ester based and ultraviolet cured-in-place pipe liners.

DOT National Transportation Integrated Search

2012-11-01

Specifications of the Virginia Department of Transportation (VDOT) allow for the use of several trenchless pipe or : culvert repair technologies whereby existing underground culverts are repaired in place rather than by the use of the conventio...

Silymarin Protects Epidermal Keratinocytes from Ultraviolet Radiation-Induced Apoptosis and DNA Damage by Nucleotide Excision Repair Mechanism

PubMed Central

Katiyar, Santosh K.; Mantena, Sudheer K.; Meeran, Syed M.

2011-01-01

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells. PMID:21731736

Properties of uvrE mutants of Escherichia coli K12. Part 1. Effects of uv irradiation on DNA metabolism

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

Vansluis, C.A.; Mattern, I.E.; Paterson, M.C.

1974-01-01

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet radiation. In an attempt to determine the underlying molecular basis for the UV sensitivity, a mutant and an isogenic wild type strain were compared with regard to several metabolic responses to 254 nm radiation. The introduction of single strand breaks into intracellular DNA after irradiation is normal; however, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain. These data suggest that the uvrEmore » gene product may be involved in a reaction between the incision and excision steps in the excision repair process. (Author) (GRA)« less

Nucleotide excision repair deficient mouse models and neurological disease

PubMed Central

Niedernhofer, Laura J.

2008-01-01

Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER deficiency. PMID:18272436

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12.

PubMed Central

Castellazzi, M; Jacques, M; George, J

1980-01-01

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent. PMID:6451614

SHINING A LIGHT ON XERODERMA PIGMENTOSUM

PubMed Central

DiGiovanna, John J.; Kraemer, Kenneth H.

2012-01-01

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disorder of DNA repair characterized by sun sensitivity and ultraviolet (UV) induced skin and mucous membrane cancers. Described in 1874 by Moriz Kaposi in Vienna, nearly 100 years later James Cleaver in San Francisco reported defective DNA repair in XP cells. This eventually provided the basis for a mechanistic link between sun exposure, DNA damage, somatic mutations and skin cancer. XP cells were found to have defects in 7 of the proteins of the nucleotide excision repair pathway and in DNA polymerase eta. XP cells are hypersensitive to killing by UV and XP cancers have characteristic “UV signature” mutations. Clinical studies at NIH found a nearly 10,000-fold increase in skin cancer in XP patients under age 20 years demonstrating the substantial importance of DNA repair in cancer prevention in the general population. About 25 % of XP patients have progressive neurological degeneration with progressive loss of neurons, probably from DNA damage induced by oxidative metabolism which kills non-dividing cells in the nervous system. Interestingly, patients with another disorder, trichothiodystrophy have defects in some of the same genes as XP but they have primary developmental abnormalities without an increase in skin cancer. PMID:22217736

Transcription coupled repair deficiency protects against human mutagenesis and carcinogenesis: Personal Reflections on the 50th anniversary of the discovery of xeroderma pigmentosum.

PubMed

Cleaver, James E

2017-10-01

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

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

Makarova, Kira S.; Omelchenko, Marina V.; Gaidamakova, Elena K.

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at itsoptimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radioduransmore » genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.« less

Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

PubMed

Son, Ji-Hye; Hwang, Eurim C; Kim, Joungmok

2016-03-01

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions.

Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice.

PubMed

Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

2016-01-01

Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student's unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. This study demonstrated the anti-aging and wound-healing effects of PPF extract. Therefore, PPF extract represents a promising new therapeutic agent for anti-aging and wound-healing treatments.

ULTRAVIOLET RADIATION AND ARSENIC INTERACTIONS: EFFECTS ON CLADOCERANS

EPA Science Inventory

The effects of arsenic and ultraviolet radiation (UV) on cladocerans have been examined separately, however the interaction of these two stresses has not been explored. Potential synergism between these two stresses is possible as arsenic is known to inhibit repair of UV induced ...

Genome Resequencing Identifies Unique Adaptations of Tibetan Chickens to Hypoxia and High-Dose Ultraviolet Radiation in High-Altitude Environments

PubMed Central

Zhang, Qian; Gou, Wenyu; Wang, Xiaotong; Zhang, Yawen; Ma, Jun; Zhang, Hongliang; Zhang, Ying; Zhang, Hao

2016-01-01

Tibetan chicken, unlike their lowland counterparts, exhibit specific adaptations to high-altitude conditions. The genetic mechanisms of such adaptations in highland chickens were determined by resequencing the genomes of four highland (Tibetan and Lhasa White) and four lowland (White Leghorn, Lindian, and Chahua) chicken populations. Our results showed an evident genetic admixture in Tibetan chickens, suggesting a history of introgression from lowland gene pools. Genes showing positive selection in highland populations were related to cardiovascular and respiratory system development, DNA repair, response to radiation, inflammation, and immune responses, indicating a strong adaptation to oxygen scarcity and high-intensity solar radiation. The distribution of allele frequencies of nonsynonymous single nucleotide polymorphisms between highland and lowland populations was analyzed using chi-square test, which showed that several differentially distributed genes with missense mutations were enriched in several functional categories, especially in blood vessel development and adaptations to hypoxia and intense radiation. RNA sequencing revealed that several differentially expressed genes were enriched in gene ontology terms related to blood vessel and respiratory system development. Several candidate genes involved in the development of cardiorespiratory system (FGFR1, CTGF, ADAM9, JPH2, SATB1, BMP4, LOX, LPR, ANGPTL4, and HYAL1), inflammation and immune responses (AIRE, MYO1F, ZAP70, DDX60, CCL19, CD47, JSC, and FAS), DNA repair, and responses to radiation (VCP, ASH2L, and FANCG) were identified to play key roles in the adaptation to high-altitude conditions. Our data provide new insights into the unique adaptations of highland animals to extreme environments. PMID:26907498

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Protective mechanisms and acclimation to solar ultraviolet-B radiation in Oenothera stricta

NASA Technical Reports Server (NTRS)

Robberecht, R.; Caldwell, M. M.

1981-01-01

Plant adaptations ameliorating or repairing the damaging effects of ultraviolet-B (UV-B) radiation on plant tissue were investigated. The degree of phenotype plasticity in UV protective mechanisms and acclimation in relation to the natural solar UV-B radiation flux and in an enhanced UV-B irradiance environment was also examined. Mechanisms by which plants avoid radiation, adaptations altering the path of radiation incident on the leaf, and repair processes were considered. Attenuation of UV-B by tissues, UV-B irradiation into the leaf, and the effects of UV-B on photosynthesis were investigated.

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

PubMed Central

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

1998-01-01

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

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

PubMed

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

1998-09-01

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

Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

PubMed Central

2016-01-01

Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions. PMID:27654267

Ultraviolet-Induced Decrease in Integration of Haemophilus influenzae Transforming Deoxyribonucleic Acid in Sensitive and Resistant Cells

PubMed Central

Muhammed, Amir; Setlow, Jane K.

1970-01-01

The decrease in integration of transforming deoxyribonucleic acid (DNA) caused by ultraviolet irradiation of the DNA was found to be independent of the presence or absence of excision repair in the recipient cell. Much of the ultraviolet-induced inhibition of integration resulted from the presence in the transforming DNA of pyrimidine dimers, as judged by the photoreactivability of the inhibition with yeast photoreactivating enzyme. The inhibition of integration made only a small contribution to the inactivation of transforming ability of the DNA by ultraviolet radiation. PMID:5308769

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

PubMed Central

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

Exposure to 4100K fluorescent light elicits sex specific transcriptional responses in Xiphophorus maculatus skin.

PubMed

Boswell, William T; Boswell, Mikki; Walter, Dylan J; Navarro, Kaela L; Chang, Jordan; Lu, Yuan; Savage, Markita G; Shen, Jianjun; Walter, Ronald B

2018-06-01

It has been reported that exposure to artificial light may affect oxygen intake, heart rate, absorption of vitamins and minerals, and behavioral responses in humans. We have reported specific gene expression responses in the skin of Xiphophorus fish after exposure to ultraviolet light (UV), as well as, both broad spectrum and narrow waveband visible light. In regard to fluorescent light (FL), we have shown that male X. maculatus exposed to 4100K FL (i.e. "cool white") rapidly suppress transcription of many genes involved with DNA replication and repair, chromosomal segregation, and cell cycle progression in skin. We have also detailed sex specific transcriptional responses of Xiphophorus skin after exposure to UVB. However, investigation of gender differences in global gene expression response after exposure to 4100K FL has not been reported, despite common use of this FL source for residential, commercial, and animal facility illumination. Here, we compare RNA-Seq results analyzed to assess changes in the global transcription profiles of female and male X. maculatus skin in response to 4100K FL exposure. Our results suggest 4100K FL exposure incites a sex-biased genetic response including up-modulation of inflammation in females and down modulation of DNA repair/replication in males. In addition, we identify clusters of genes that become oppositely modulated in males and females after FL exposure that are principally involved in cell death and cell proliferation. Copyright © 2017 Elsevier Inc. All rights reserved.

Endonuclease from Micrococcus luteus Which Has Activity Toward Ultraviolet-Irradiated Deoxyribonucleic Acid: Its Action on Transforming Deoxyribonucleic Acid

PubMed Central

Setlow, R. B.; Setlow, Jane K.; Carrier, W. L.

1970-01-01

An endonuclease purified from Micrococcus luteus makes single-strand breaks in ultraviolet (UV)-irradiated, native deoxyribonucleic acid (DNA). The purified endonuclease is able to reactivate UV-inactivated transforming DNA of Haemophilus influenzae, especially when the DNA is assayed on a UV-sensitive mutant of H. influenzae. After extensive endonuclease action, there is a loss of transforming DNA when assayed on both UV-sensitive and -resistant cells. The endonuclease does not affect unirradiated DNA. The results indicate that the endonuclease function is involved in the repair of biological damage resulting from UV irradiation and that the UV-sensitive mutant is deficient in this step. We interpret the data as indicating that the various steps in the repair of DNA must be well coordinated if repair is to be effective. PMID:4314478

Photocarcinogenesis and Skin Cancer Prevention Strategies.

PubMed

Seebode, Christina; Lehmann, Janin; Emmert, Steffen

2016-03-01

In this review the basic principles of UV-induced carcinogenesis are summarized and the state of the art diagnosis and therapeutic strategies are discussed. The prevalent keratinocyte-derived neoplasms of the skin are basal cell and squamous cell carcinomas. Cutaneous melanoma is less frequent but associated with high mortality. Common risk factors for all three tumor entities include sun exposure and DNA-repair deficiencies. Photocarcinogenesis follows a multistep model of cancer development in which ultraviolet-induced DNA damage leads to mutations resulting in activation of oncogenes or silencing of tumor-suppressor genes. This ends in a cellular mutator phenotype even more prone to mutation acquisition. DNA repair, especially the nucleotide excision repair (NER) pathway, counteracts mutation formation and skin cancer development. This is vividly demonstrated by the NER-defective disorder xeroderma pigmentosum. Primary skin cancer preventative strategies, therefore, include reduction of DNA photodamage by protection from the sun. Secondary preventative strategies include skin cancer screening. This implies standard examination techniques with the naked eye, an epiluminescence microscope, or digital epiluminescence microscopy. More advanced techniques include confocal laser scan microscopy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Genome Resequencing Identifies Unique Adaptations of Tibetan Chickens to Hypoxia and High-Dose Ultraviolet Radiation in High-Altitude Environments.

PubMed

Zhang, Qian; Gou, Wenyu; Wang, Xiaotong; Zhang, Yawen; Ma, Jun; Zhang, Hongliang; Zhang, Ying; Zhang, Hao

2016-02-23

Tibetan chicken, unlike their lowland counterparts, exhibit specific adaptations to high-altitude conditions. The genetic mechanisms of such adaptations in highland chickens were determined by resequencing the genomes of four highland (Tibetan and Lhasa White) and four lowland (White Leghorn, Lindian, and Chahua) chicken populations. Our results showed an evident genetic admixture in Tibetan chickens, suggesting a history of introgression from lowland gene pools. Genes showing positive selection in highland populations were related to cardiovascular and respiratory system development, DNA repair, response to radiation, inflammation, and immune responses, indicating a strong adaptation to oxygen scarcity and high-intensity solar radiation. The distribution of allele frequencies of nonsynonymous single nucleotide polymorphisms between highland and lowland populations was analyzed using chi-square test, which showed that several differentially distributed genes with missense mutations were enriched in several functional categories, especially in blood vessel development and adaptations to hypoxia and intense radiation. RNA sequencing revealed that several differentially expressed genes were enriched in gene ontology terms related to blood vessel and respiratory system development. Several candidate genes involved in the development of cardiorespiratory system (FGFR1, CTGF, ADAM9, JPH2, SATB1, BMP4, LOX, LPR, ANGPTL4, and HYAL1), inflammation and immune responses (AIRE, MYO1F, ZAP70, DDX60, CCL19, CD47, JSC, and FAS), DNA repair, and responses to radiation (VCP, ASH2L, and FANCG) were identified to play key roles in the adaptation to high-altitude conditions. Our data provide new insights into the unique adaptations of highland animals to extreme environments. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts

PubMed Central

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-01-01

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

PubMed

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-11-24

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.

Apigenin prevents ultraviolet-B radiation induced cyclobutane pyrimidine dimers formation in human dermal fibroblasts.

PubMed

Britto, S Mary; Shanthakumari, D; Agilan, B; Radhiga, T; Kanimozhi, G; Prasad, N Rajendra

2017-09-01

Exposure to solar ultraviolet-B (UVB) radiation leads to the formation of cyclobutane pyrimidine dimers (CPDs). We investigated the protective effect of apigenin against UVB-induced CPDs formation in human dermal fibroblasts cells (HDFa). For this purpose, HDFa cells were treated with apigenin (15μM) prior to UVB irradiation (20mJ/cm 2 ); DNA damage and subsequent molecular end points were observed. Exposure to UVB radiation increased significant CPDs formation in HDFa cells and the frequencies of CPDs were reduced by treatment with apigenin (15μM). UVB-induced CPDs downregulates the expression of nucleotide excision repair (NER) genes such as xeroderma pigmentosum complementation group C, B, G and F (XPC, XPB, XPG and XPF), transcription factor II human (TFIIH) and excision repair cross-complementation group 1 (ERCC1) in HDFa cells. Conversely, apigenin treatment restored UVB-induced loss of NER proteins in HDFa cells, which indicates its preventive effect against CPDs formation. Besides, single low dose UVB-exposure induced nuclear fragmentation, apoptotic frequency and apoptotic proteins expression (Bax and Caspase-3) have been prevented by the apigenin pretreatment. Furthermore, apigenin exhibits strong UV absorbance property and showed 10.08 SPF value. Thus, apigenin can protect skin cells against UVB-induced CPDs formation probably through its sunscreen effect. Hence, apigenin can be considered as an effective protective agent against UV induced skin damages. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of space environment on T-7 bacteriophage and spores of Bacillus subtilis 168

NASA Technical Reports Server (NTRS)

Spizizen, J.; Isherwood, J. E.

1973-01-01

Two strains of Bacillus subtilis were exposed to components of the ultraviolet spectrum in space. Both strains possess multiple genetic markers, and one of the strains is defective in the ability to repair ultraviolet damage. The T-7 bacteriophage of Escherichia coli was also exposed to selected wavelengths and energy levels of ultraviolet light in space. Preliminary findings do not reveal anomalies in survival rates. Data are not yet available on detailed genetic analyses.

UV Radiation Damage and Bacterial DNA Repair Systems

ERIC Educational Resources Information Center

Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

2006-01-01

This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

COMPARISON OF UV INACTIVATION OF SPORES OF THREE ENCEPHALITOZOON SPECIES WITH THAT OF SPORES OF TWO DNA REPAIR-DEFICIENT BACILLUS SUBTILIS BIODOSIMETRY STRAINS

EPA Science Inventory

The sensitivity of three Encephalitozoon spp. to ultraviolet (UV) inactivation was determined. Encephalitozoon intestinalis is a contaminant listed on the USEPA's 1998 Contaminant Candidate List (CCL). Also, use of DNA repair deficient strains of Bacillus subtilis were evaluat...

Translations on USSR Science and Technology, Biomedical and Behavioral Sciences, Number 39

DTIC Science & Technology

1978-07-28

hereditary impairment of repair processes in cells of the human body leads to such serious diseases as xeroderma pigmentosum , which is due to the...inability of skin fibroblasts to repair DNA lesions induced by ultraviolet light or x-rays. In addition to xeroderma pigmentosum , Bloom’s syndrome

Ultraviolet laser ablation as technique for defect repair of GaN-based light-emitting diodes

NASA Astrophysics Data System (ADS)

Passow, Thorsten; Kunzer, Michael; Pfeuffer, Alexander; Binder, Michael; Wagner, Joachim

2018-03-01

Defect repair of GaN-based light-emitting diodes (LEDs) by ultraviolet laser micromachining is reported. Percussion and helical drilling in GaN by laser ablation were investigated using 248 nm nanosecond and 355 nm picosecond pulses. The influence of laser ablation including different laser parameters on electrical and optical properties of GaN-based LED chips was evaluated. The results for LEDs on sapphire with transparent conductive oxide p-type contact on top as well as for thin-film LEDs are reported. A reduction of leakage current by up to six orders in magnitude and homogeneous luminance distribution after proper laser defect treatment were achieved.

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

PubMed Central

Sorokin, Dmitry V; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, Pavel; Kozubek, Stanislav; Matula, Pavel; Skalníková, Magdalena; Raška, Ivan; Bártová, Eva

2015-01-01

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase. PMID:26208041

Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice

PubMed Central

Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

2016-01-01

Background Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Objective Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. Methods PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student’s unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. Results PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. Conclusion This study demonstrated the anti-aging and wound-healing effects of PPF extract. Therefore, PPF extract represents a promising new therapeutic agent for anti-aging and wound-healing treatments. PMID:27536082

Photodamage: all signs lead to actinic keratosis and early squamous cell carcinoma.

PubMed

Wei, Jerry; Kok, Lai Fong; Byrne, Scott N; Halliday, Gary M

2015-01-01

Ultraviolet (UV) radiation is likely to drive the initiation and progression of skin cancer from actinic keratosis to squamous cell carcinoma. Signs of photodamage occur at multiple steps. UV radiation damages many cellular constituents, including lipids, proteins and DNA, all of which are likely to contribute to UV-induced skin cancer. Two biological events culminating from photodamage are mutations in the genes critical to the control of cell division, differentiation and invasion and immunosuppression. DNA photodamage, if unrepaired prior to cell division, can result in the incorporation of an incorrect nucleotide into newly synthesised DNA. Mutations in critical genes contribute to carcinogenesis. Photodamage to proteins such as those involved in DNA repair or proteins or lipids involved in cellular signalling can interfere with this repair process and contribute to mutagenesis. Mutations in key genes, including TP53, BRM, PTCH1, and HRAS, contribute to skin carcinogenesis. UV also damages immunity. Photodamage to DNA and signalling lipids as well as other molecular changes are detrimental to the key cells that regulate immunity. Photodamaged dendritic cells and altered responses by mast cells lead to the activation of T and B regulatory cells that suppress immunity to the protein products of UV-mutated genes. This stops the immune response from its protective function of destroying mutated cells, enabling the transformed cells to progress to skin cancer. UV appears to play a pivotal role at each of these steps, and therefore, signs of photodamage point to the development of skin cancer. © 2015 S. Karger AG, Basel.

A genome-wide analysis of gene–caffeine consumption interaction on basal cell carcinoma

PubMed Central

Li, Xin; Cornelis, Marilyn C.; Liang, Liming; Song, Fengju; De Vivo, Immaculata; Giovannucci, Edward; Tang, Jean Y.; Han, Jiali

2016-01-01

Animal models have suggested that oral or topical administration of caffeine could inhibit ultraviolet-induced carcinogenesis via the ataxia telangiectasia and rad3 (ATR)-related apoptosis. Previous epidemiological studies have demonstrated that increased caffeine consumption is associated with reduced risk of basal cell carcinoma (BCC). To identify common genetic markers that may modify this association, we tested gene–caffeine intake interaction on BCC risk in a genome-wide analysis. We included 3383 BCC cases and 8528 controls of European ancestry from the Nurses’ Health Study and Health Professionals Follow-up Study. Single nucleotide polymorphism (SNP) rs142310826 near the NEIL3 gene showed a genome-wide significant interaction with caffeine consumption (P = 1.78 × 10–8 for interaction) on BCC risk. There was no gender difference for this interaction (P = 0.64 for heterogeneity). NEIL3, a gene belonging to the base excision DNA repair pathway, encodes a DNA glycosylase that recognizes and removes lesions produced by oxidative stress. In addition, we identified several loci with P value for interaction <5 × 10–7 in gender-specific analyses (P for heterogeneity between genders < 0.001) including those mapping to the genes LRRTM4, ATF3 and DCLRE1C in women and POTEA in men. Finally, we tested the associations between caffeine consumption-related SNPs reported by previous genome-wide association studies and risk of BCC, both individually and jointly, but found no significant association. In sum, we identified a DNA repair gene that could be involved in caffeine-mediated skin tumor inhibition. Further studies are warranted to confirm these findings. PMID:27797824

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea

PubMed Central

Jones, Daniel L.; Baxter, Bonnie K.

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a “first line of defense,” and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms. PMID:29033920

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea.

PubMed

Jones, Daniel L; Baxter, Bonnie K

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a "first line of defense," and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms.

Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511

PubMed Central

2010-01-01

Background The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (~1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation. Results The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure. Conclusions Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes. PMID:20670397

Temporal variations in sirtuin expression under normal and ultraviolet B-induced conditions and their correlation to energy levels in normal human epidermal keratinocytes.

PubMed

Pelle, Edward; Dong, Kelly; Pernodet, Nadine

2015-01-01

Sirtuins are post-translational modifiers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identified as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the temporal variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sirtuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. Moreover, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show significant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB-induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

PubMed

McLean, K M; Gutman, P D; Minton, K W; Clark, E P

1992-06-01

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

PubMed

Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

2016-04-06

Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

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

Stark, M.; Naiman, T.; Canaani, D.

1989-08-15

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

Gene Therapy for Cartilage Repair

PubMed Central

Madry, Henning; Orth, Patrick; Cucchiarini, Magali

2011-01-01

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

DNA damage and repair in plants under ultraviolet and ionizing radiations.

PubMed

Gill, Sarvajeet S; Anjum, Naser A; Gill, Ritu; Jha, Manoranjan; Tuteja, Narendra

2015-01-01

Being sessile, plants are continuously exposed to DNA-damaging agents present in the environment such as ultraviolet (UV) and ionizing radiations (IR). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315-400 nm; UV-B, 280-315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damage. On the other hand, IR causes water radiolysis, which generates highly reactive hydroxyl radicals (OH(•)) and causes radiogenic damage to important cellular components. However, to maintain genomic integrity under UV/IR exposure, plants make use of several DNA repair mechanisms. In the light of recent breakthrough, the current minireview (a) introduces UV/IR and overviews UV/IR-mediated DNA damage products and (b) critically discusses the biochemistry and genetics of major pathways responsible for the repair of UV/IR-accrued DNA damage. The outcome of the discussion may be helpful in devising future research in the current context.

Xeroderma pigmentosum neurological abnormalities correlate with colony-forming ability after ultraviolet radiation

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

Andrews, A.D.; Barrett, S.F.; Robbins, J.H.

1978-04-01

Xeroderma pigmentosum is an autosomal recessive disease in which DNA repair processes are defective. All xeroderma pigmentosum patients develop premature aging of sun exposed skin, and some develop neurological abnormalities due to premature death of nerve cells. Sensitivity to ultraviolet radiation of 24 xeroderma pigmentosum fibroblast strains was studied in vitro by measuring each strain's ability to divide and form colonies after irradiation. The most sensitive strains were derived from patients who had an early onset of neurological abnormalities; less sensitive strains were from patients with a later onset; and the most resistant strains were from patients without neurological abnormalities.more » The uv sensitivities of strains from each member of a sibling pair with xeroderma pigmentosum were identical, indicating that uv sensitivity of xeroderma pigmentosum strains is determined by the patient's inherited DNA repair defect. The results suggest that effective DNA repair is required to maintain the functional integrity of the human nervous system by preventing premature death of neurons.« less

Repair of localized defects in multilayer-coated reticle blanks for extreme ultraviolet lithography

DOEpatents

Stearns, Daniel G [Los Altos, CA; Sweeney, Donald W [San Ramon, CA; Mirkarimi, Paul B [Sunol, CA

2004-11-23

A method is provided for repairing defects in a multilayer coating layered onto a reticle blank used in an extreme ultraviolet lithography (EUVL) system. Using high lateral spatial resolution, energy is deposited in the multilayer coating in the vicinity of the defect. This can be accomplished using a focused electron beam, focused ion beam or a focused electromagnetic radiation. The absorbed energy will cause a structural modification of the film, producing a localized change in the film thickness. The change in film thickness can be controlled with sub-nanometer accuracy by adjusting the energy dose. The lateral spatial resolution of the thickness modification is controlled by the localization of the energy deposition. The film thickness is adjusted locally to correct the perturbation of the reflected field. For example, when the structural modification is a localized film contraction, the repair of a defect consists of flattening a mound or spreading out the sides of a depression.

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems.

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

PubMed Central

Mogi, Seiki; Oh, Dennis H.

2009-01-01

To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (γ-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, γ-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced γ-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited γ-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce γ-H2AX in XP-F cells, although it did induce equivalent levels of γ-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored γ-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of γ-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form γ-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate. PMID:16678501

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4.

PubMed

Chenevert, J M; Naumovski, L; Schultz, R A; Friedberg, E C

1986-04-01

The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.

Ophthalmic manifestations and histopathology of xeroderma pigmentosum: two clinicopathological cases and a review of the literature.

PubMed

Ramkumar, Hema L; Brooks, Brian P; Cao, Xiaoguang; Tamura, Deborah; Digiovanna, John J; Kraemer, Kenneth H; Chan, Chi-Chao

2011-01-01

Xeroderma pigmentosum is a rare, autosomal recessive disease caused by a defect in DNA repair. Patients with xeroderma pigmentosum often have cutaneous and ocular sun sensitivity, freckle-like skin pigmentation, multiple skin and eye cancers, and, in some patients, progressive neurodegeneration. Xeroderma pigmentosum predominantly affects the ultraviolet (UV) exposed ocular surface, resulting in eyelid atrophy and cancers, corneal dryness, exposure keratopathy, and conjunctival tumors. We report the clinical history and ocular pathology of two white women who had xeroderma pigmentosum with neurological degeneration: Case 1 (died at age 44 years) and Case 2 (died at age 45 years). Case 1, with mutations in the XPA gene, had more than 180 basal cell carcinomas of her skin and eyelids and died from complications of neurodegeneration. Case 2, with mutations in the XPD gene, was sun-protected and had three skin cancers. She died from complications of neurodegeneration and pneumonia. Both patients had bilateral pinguecula, corneal pannus, and exposure keratopathy. Case 1 had bilateral optic atrophy, and Case 2 had bilateral peripheral retinal pigmentary degeneration. Both patients developed retinal gliosis. The ophthalmic manifestations and pathology of xeroderma pigmentosum are discussed and reviewed with respect to this report and other cases in the literature. These cases illustrate the role of DNA repair in protection of the eyes from UV damage and neurodegeneration of the retina. Published by Elsevier Inc.

Preclinical Corrective Gene Transfer in Xeroderma Pigmentosum Human Skin Stem Cells

PubMed Central

Warrick, Emilie; Garcia, Marta; Chagnoleau, Corinne; Chevallier, Odile; Bergoglio, Valérie; Sartori, Daniela; Mavilio, Fulvio; Angulo, Jaime F; Avril, Marie-Françoise; Sarasin, Alain; Larcher, Fernando; Del Rio, Marcela; Bernerd, Françoise; Magnaldo, Thierry

2012-01-01

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>1040 cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients. PMID:22068429

Light-Curing Adhesive Repair Tapes

NASA Technical Reports Server (NTRS)

Allred, Ronald; Haight, Andrea Hoyt

2009-01-01

Adhesive tapes, the adhesive resins of which can be cured (and thereby rigidized) by exposure to ultraviolet and/or visible light, are being developed as repair patch materials. The tapes, including their resin components, consist entirely of solid, low-outgassing, nonhazardous or minimally hazardous materials. They can be used in air or in vacuum and can be cured rapidly, even at temperatures as low as -20 C. Although these tapes were originally intended for use in repairing structures in outer space, they can also be used on Earth for quickly repairing a wide variety of structures. They can be expected to be especially useful in situations in which it is necessary to rigidize tapes after wrapping them around or pressing them onto the parts to be repaired.

Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

PubMed Central

Kachhap, Sushant K.; Rosmus, Nadine; Collis, Spencer J.; Kortenhorst, Madeleine S. Q.; Wissing, Michel D.; Hedayati, Mohammad; Shabbeer, Shabana; Mendonca, Janet; Deangelis, Justin; Marchionni, Luigi; Lin, Jianqing; Höti, Naseruddin; Nortier, Johan W. R.; DeWeese, Theodore L.; Hammers, Hans; Carducci, Michael A.

2010-01-01

Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics. PMID:20585447

Basal cell carcinoma, squamous cell carcinoma and melanoma of the head and face.

PubMed

Feller, L; Khammissa, R A G; Kramer, B; Altini, M; Lemmer, J

2016-02-05

Ultraviolet light (UV) is an important risk factor for cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin. These cancers most commonly affect persons with fair skin and blue eyes who sunburn rather than suntan. However, each of these cancers appears to be associated with a different pattern of UV exposure and to be mediated by different intracellular molecular pathways.Some melanocortin 1 receptor (MC1R) gene variants play a direct role in the pathogenesis of cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma apart from their role in determining a cancer-prone pigmentory phenotype (fair skin, red hair, blue eyes) through their interactions with other genes regulating immuno-inflammatory responses, DNA repair or apoptosis.In this short review we focus on the aetiological role of UV in cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin, and on some associated biopathological events.

Acetylation of hMOF Modulates H4K16ac to Regulate DNA Repair Genes in Response to Oxidative Stress.

PubMed

Zhong, Jianing; Ji, Liying; Chen, Huiqian; Li, Xianfeng; Zhang, Jian'an; Wang, Xingxing; Wu, Weilin; Xu, Ying; Huang, Fei; Cai, Wanshi; Sun, Zhong Sheng

2017-01-01

Oxidative stress is considered to be a key risk state for a variety of human diseases. In response to oxidative stress, the regulation of transcriptional expression of DNA repair genes would be important to DNA repair and genomic stability. However, the overall pattern of transcriptional expression of DNA repair genes and the underlying molecular response mechanism to oxidative stress remain unclear. Here, by employing colorectal cancer cell lines following exposure to hydrogen peroxide, we generated expression profiles of DNA repair genes via RNA-seq and identified gene subsets that are induced or repressed following oxidative stress exposure. RRBS-seq analyses further indicated that transcriptional regulation of most of the DNA repair genes that were induced or repressed is independent of their DNA methylation status. Our analyses also indicate that hydrogen peroxide induces deacetylase SIRT1 which decreases chromatin affinity and the activity of histone acetyltransferase hMOF toward H4K16ac and results in decreased transcriptional expression of DNA repair genes. Taken together, our findings provide a potential mechanism by which oxidative stress suppresses DNA repair genes which is independent of the DNA methylation status of their promoters.

DNA Damage and Repair in Plants under Ultraviolet and Ionizing Radiations

PubMed Central

Gill, Sarvajeet S.; Gill, Ritu; Jha, Manoranjan; Tuteja, Narendra

2015-01-01

Being sessile, plants are continuously exposed to DNA-damaging agents present in the environment such as ultraviolet (UV) and ionizing radiations (IR). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315–400 nm; UV-B, 280–315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damage. On the other hand, IR causes water radiolysis, which generates highly reactive hydroxyl radicals (OH•) and causes radiogenic damage to important cellular components. However, to maintain genomic integrity under UV/IR exposure, plants make use of several DNA repair mechanisms. In the light of recent breakthrough, the current minireview (a) introduces UV/IR and overviews UV/IR-mediated DNA damage products and (b) critically discusses the biochemistry and genetics of major pathways responsible for the repair of UV/IR-accrued DNA damage. The outcome of the discussion may be helpful in devising future research in the current context. PMID:25729769

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

PubMed

Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

2006-08-30

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

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

Alexandrov, L. B.

All cancers originate from a single cell that starts to behave abnormally, to divide uncontrollably, and, eventually, to invade adjacent tissues (1). The aberrant behavior of this single cell is due to somatic mutations—changes in the genomic DNA produced by the activity of different mutational processes (1). These various mutational processes include exposure to exogenous or endogenous mutagens, abnormal DNA editing, the incomplete fidelity of DNA polymerases, and failure of DNA repair mechanisms (2). Early studies that sequenced TP53, the most commonly mutated gene in human cancer, provided evidence that mutational processes leave distinct imprints of somatic mutations on themore » genome of a cancer cell (3). For example, C:G>A:T transversions predominate in smoking-associated lung cancer, whereas C:G>T:A transitions occurring mainly at dipyrimidines and CC:GG>TT:AA double-nucleotide substitutions are common in ultraviolet light–associated skin cancers. Moreover, these patterns of mutations matched the ones induced experimentally by tobacco mutagens and ultraviolet light, respectively, the major, known, exogenous carcinogenic influences in these cancer types, and demonstrated that examining patterns of mutations in cancer genomes can yield information about the mutational processes that cause human cancer (4).« less

Understanding the origins of human cancer

DOE PAGES

Alexandrov, L. B.

2015-12-04

All cancers originate from a single cell that starts to behave abnormally, to divide uncontrollably, and, eventually, to invade adjacent tissues (1). The aberrant behavior of this single cell is due to somatic mutations—changes in the genomic DNA produced by the activity of different mutational processes (1). These various mutational processes include exposure to exogenous or endogenous mutagens, abnormal DNA editing, the incomplete fidelity of DNA polymerases, and failure of DNA repair mechanisms (2). Early studies that sequenced TP53, the most commonly mutated gene in human cancer, provided evidence that mutational processes leave distinct imprints of somatic mutations on themore » genome of a cancer cell (3). For example, C:G>A:T transversions predominate in smoking-associated lung cancer, whereas C:G>T:A transitions occurring mainly at dipyrimidines and CC:GG>TT:AA double-nucleotide substitutions are common in ultraviolet light–associated skin cancers. Moreover, these patterns of mutations matched the ones induced experimentally by tobacco mutagens and ultraviolet light, respectively, the major, known, exogenous carcinogenic influences in these cancer types, and demonstrated that examining patterns of mutations in cancer genomes can yield information about the mutational processes that cause human cancer (4).« less

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

PubMed

Martin, P; Prakash, L; Prakash, S

1981-05-01

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA.

PubMed Central

Venema, J; van Hoffen, A; Natarajan, A T; van Zeeland, A A; Mullenders, L H

1990-01-01

We have measured removal of pyrimidine dimers in defined DNA sequences in confluent and actively growing normal human and xeroderma pigmentosum complementation group C (XP-C) fibroblasts exposed to 10 J/m2 UV-irradiation. In normal fibroblasts 45% and 90% of the dimers are removed from the transcriptionally active adenosine deaminase (ADA) gene within 4 and 24 hours after irradiation respectively. Equal repair efficiencies are found in fragments located entirely within the transcription unit or partly in the 3' flanking region of the ADA gene. The rate and extent of dimer removal from the dihydrofolate reductase (DHFR) gene is very similar to that of the ADA gene. Repair of the transcriptionally inactive 754 locus is less efficient: 18% and 52% of the dimers are removed within 4 and 24 hours respectively. In spite of the limited overall repair capacity, confluent XP-C fibroblasts efficiently remove dimers from the ADA and DHFR genes: about 90% and 50% within 24 hours respectively. The 3' end of the ADA gene is repaired as efficiently as in normal human fibroblasts, but less efficient repair occurs in DNA fragments located in the DHFR gene and at the 5' end of the ADA gene. Repair of the inactive 754 locus does not exceed the very slow rate of dimer removal from the genome overall. Confluent and actively growing XP-C cells show similar efficiencies of repair of the ADA, DHFR and 754 genes. Our findings suggest the existence of two independently operating pathways directed towards repair of pyrimidine dimers in either active or inactive chromatin. XP-C cells have lost the capacity to repair inactive chromatin, but are still able to repair active chromatin. Images PMID:2308842

Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

PubMed Central

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

2015-01-01

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

The Cell's Sophisticated Army to Defend Against Assaults on DNAThe Cell's Sophisticated Army to Defend Against Assaults on DNA | Center for Cancer Research

Cancer.gov

The maintenance of genome integrity and function is essen-tial for the survival of cells and organisms. Any damage to our genetic material must be immediately sensed and repaired to preserve a cell’s func-tional integrity. Cells are constantly faced with the challenge of protecting their DNA from assaults by damaging chemicals and ultraviolet light. DNA damage that escapes repair can lead to a variety of genetic disorders and diseases, particularly cancer. To avoid this catastrophe, the cell employs an army of DNA repair factors that “rush to the scene” and initiate a cascade of events to repair the damage. Exactly how different repair factors sense DNA damage and orchestrate their concert-ed response is not well understood.

Nicotinamide Enhances Repair of Arsenic and Ultraviolet Radiation-Induced DNA Damage in HaCaT Keratinocytes and Ex Vivo Human Skin

PubMed Central

Thompson, Benjamin C.; Halliday, Gary M.; Damian, Diona L.

2015-01-01

Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2′-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer. PMID:25658450

DNA Excision Repair at Telomeres

PubMed Central

Jia, Pingping; Her, Chengtao; Chai, Weihang

2015-01-01

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance. PMID:26422132

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

NASA Technical Reports Server (NTRS)

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

2008-01-01

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

Xeroderma pigmentosum.

PubMed

Lehmann, Alan R; McGibbon, David; Stefanini, Miria

2011-11-01

Xeroderma pigmentosum (XP) is defined by extreme sensitivity to sunlight, resulting in sunburn, pigment changes in the skin and a greatly elevated incidence of skin cancers. It is a rare autosomal recessive disorder and has been found in all continents and racial groups. Estimated incidences vary from 1 in 20, 000 in Japan to 1 in 250, 000 in the USA, and approximately 2.3 per million live births in Western Europe.The first features are either extreme sensitivity to sunlight, triggering severe sunburn, or, in patients who do not show this sun-sensitivity, abnormal lentiginosis (freckle-like pigmentation due to increased numbers of melanocytes) on sun-exposed areas. This is followed by areas of increased or decreased pigmentation, skin aging and multiple skin cancers, if the individuals are not protected from sunlight. A minority of patients show progressive neurological abnormalities. There are eight XP complementation groups, corresponding to eight genes, which, if defective, can result in XP. The products of these genes are involved in the repair of ultraviolet (UV)-induced damage in DNA. Seven of the gene products (XPA through G) are required to remove UV damage from the DNA. The eighth (XPV or DNA polymerase η) is required to replicate DNA containing unrepaired damage. There is wide variability in clinical features both between and within XP groups. Diagnosis is made clinically by the presence, from birth, of an acute and prolonged sunburn response at all exposed sites, unusually early lentiginosis in sun-exposed areas or onset of skin cancers at a young age. The clinical diagnosis is confirmed by cellular tests for defective DNA repair. These features distinguish XP from other photodermatoses such as solar urticaria and polymorphic light eruption, Cockayne Syndrome (no pigmentation changes, different repair defect) and other lentiginoses such as Peutz-Jeghers syndrome, Leopard syndrome and Carney complex (pigmentation not sun-associated), which are inherited in an autosomal dominant fashion. Antenatal diagnosis can be performed by measuring DNA repair or by mutation analysis in CVS cells or in amniocytes. Although there is no cure for XP, the skin effects can be minimised by rigorous protection from sunlight and early removal of pre-cancerous lesions. In the absence of neurological problems and with lifetime protection against sunlight, the prognosis is good. In patients with neurological problems, these are progressive, leading to disabilities and a shortened lifespan.

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

PubMed

Gupta, Rakesh Kumar; Bajpai, Deepti; Singh, Neeta

2015-01-01

Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. SiHa cells were cultured and treated with 10% Noni, 10 μg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

Persistence of Repair Proteins at Unrepaired DNA Damage Distinguishes Diseases with ERCC2 (XPD) Mutations: Cancer-Prone Xeroderma Pigmentosum vs. Non-Cancer-Prone Trichothiodystrophy

PubMed Central

Boyle, Jennifer; Ueda, Takahiro; Oh, Kyu-Seon; Imoto, Kyoko; Tamura, Deborah; Jagdeo, Jared; Khan, Sikandar G.; Nadem, Carine; DiGiovanna, John J.; Kraemer, Kenneth H.

2012-01-01

Patients with xeroderma pigmentosum (XP) have a 1,000-fold increase in ultraviolet (UV)-induced skin cancers while trichothiodystrophy (TTD) patients, despite mutations in the same genes, ERCC2 (XPD) or ERCC3 (XPB), are cancer-free. Unlike XP cells, TTD cells have a nearly normal rate of removal of UV-induced 6-4 photoproducts (6-4PP) in their DNA and low levels of the basal transcription factor, TFIIH. We examined seven XP, TTD, and XP/TTD complex patients and identified mutations in the XPD gene. We discovered large differences in nucleotide excision repair (NER) protein recruitment to sites of localized UV damage in TTD cells compared to XP or normal cells. XPC protein was rapidly localized in all cells. XPC was redistributed in TTD, and normal cells by 3 hr postirradiation, but remained localized in XP cells at 24-hr postirradiation. In XP cells recruitment of other NER proteins (XPB, XPD, XPG, XPA, and XPF) was also delayed and persisted at 24 hr (p < 0.001). In TTD cells with defects in the XPD, XPB, or GTF2H5 (TTDA) genes, in contrast, recruitment of these NER proteins was reduced compared to normals at early time points (p < 0.001) and remained low at 24 hr postirradiation. These data indicate that in XP persistence of NER proteins at sites of unrepaired DNA damage is associated with greatly increased skin cancer risk possibly by blockage of translesion DNA synthesis. In contrast, in TTD, low levels of unstable TFIIH proteins do not accumulate at sites of unrepaired photoproducts and may permit normal translesion DNA synthesis without increased skin cancer. PMID:18470933

Electron Transfer Mechanisms of DNA Repair by Photolyase

NASA Astrophysics Data System (ADS)

Zhong, Dongping

2015-04-01

Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

Dietary proanthocyanidins prevent ultraviolet radiation-induced non-melanoma skin cancer through enhanced repair of damaged DNA-dependent activation of immune sensitivity.

PubMed

Katiyar, Santosh K; Pal, Harish C; Prasad, Ram

2017-10-01

Numerous plant products have been used to prevent and manage a wide variety of diseases for centuries. These products are now considered as promising options for the development of more effective and less toxic alternatives to the systems of medicine developed primarily in developed countries in the modern era. Grape seed proanthocyanidins (GSPs) are of great interest due to their anti-carcinogenic effects that have been demonstrated using various tumor models including ultraviolet (UV) radiation-induced non-melanoma skin cancer. In a pre-clinical mouse model supplementation of a control diet (AIN76A) with GSPs at concentrations of 0.2% and 0.5% (w/w) significantly inhibits the growth and multiplicity of UVB radiation-induced skin tumors. In this review, we summarize the evidence that this inhibition of UVB-induced skin tumor development by dietary GSPs is mediated by a multiplicity of coordinated effects including: (i) Promotion of the repair of damaged DNA by nuclear excision repair mechanisms, and (ii) DNA repair-dependent stimulation of the immune system following the functional activation of dendritic cells and effector T cells. Dietary GSPs hold promise for the development of an effective alternative strategy for the prevention of excessive solar UVB radiation exposure-induced skin diseases including the risk of non-melanoma skin cancer in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

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

Intine, R.V.; Rainbow, A.J.

1990-01-01

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in partmore » at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.« less

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

PubMed

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

2016-04-01

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

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

PubMed

Jasin, Maria; Haber, James E

2016-08-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

PubMed Central

Jasin, Maria; Haber, James E.

2017-01-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

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

PubMed

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

2009-06-01

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

The production and repair of aflatoxin B sub 1 -induced DNA damage

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

Leadon, S.A.

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of aflatoxin B{sub 1} (AFB{sub 1})-induced damage in the nontranscribed, heterochromatic alpha DNA of monkey cells and in the metallothionein genes of human cells. In confluent cells, AFB{sub 1} adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha DNA is severely deficient, however, removal of AFB{sub 1} adducts from alpha DNA is enhanced by small doses of UV. The repair deficiencies are not observed in actively growing cells. We havemore » also shown that there is preferential repair of AFB{sub 1} damage in active genes. AFB{sub 1} damage is efficiently repaired in the active human metallothionein (hMT) genes, but deficiently repaired in inactive hMT genes. 51 refs., 3 tabs.« less

A genetic cluster of patients with variant xeroderma pigmentosum with two different founder mutations.

PubMed

Munford, V; Castro, L P; Souto, R; Lerner, L K; Vilar, J B; Quayle, C; Asif, H; Schuch, A P; de Souza, T A; Ienne, S; Alves, F I A; Moura, L M S; Galante, P A F; Camargo, A A; Liboredo, R; Pena, S D J; Sarasin, A; Chaibub, S C; Menck, C F M

2017-05-01

Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe. © 2016 British Association of Dermatologists.

Altered DNA Methylation and Expression Profiles of 8-Oxoguanine DNA Glycosylase 1 in Lens Tissue from Age-related Cataract Patients.

PubMed

Wang, Yong; Li, Fei; Zhang, Guowei; Kang, Lihua; Qin, Bai; Guan, Huaijin

2015-01-01

Oxidative stress and DNA damage contribute to the pathogenesis of age-related cataract (ARC). Most oxidative DNA lesions are repaired via the base excision repair (BER) proteins including 8-oxoguanine DNA glycosylase 1 (OGG1). This study examined DNA methylation of CpG islands upstream of OGG1 and their relation to the gene expression in lens cortex from ARC patients. The clinical case-control study consisted of 15 cortical type of ARC patients and 15 age-matched non-ARC controls who received transparent lens extraction due to vitreoretinal diseases. OGG1 expression in lens cortex was analyzed by qRT-PCR and Western blot. The localization and the proportion of cells positive for OGG1 were determined by immunofluorescence. Bisulfite-sequencing PCR (BSP) was performed to evaluate the methylation status of CpG islands near OGG1 in DNA extracted from lens cortex. To test relationship between the methylation and the expression of the gene of interest, 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to induce demethylation of cultured human lens epithelium B-3 (HLE B-3). To test the role of OGG1 in the repair of cellular damage, HLE B-3 was transfected with OGG1 vector, followed by ultraviolet radiation b (UVB) exposure to induce apoptosis. The mRNA and protein levels of OGG1 were significantly reduced in the lens cortex of ARC. Immunofluorescence showed that the proportion of OGG1-positive cells decreased significantly in ARC cortex in comparison with the control. The CpG island in first exon of OGG1 displayed hypermethylation in the DNA extracted from the lens cortex of ARC. Treatment of HLEB-3 cells with 5-Aza-dC upregulated OGG1 expression. UVB-induced apoptosis was attenuated after transfection with OGG1. A reduced OGG1 expression was correlated with hypermethylation of a CpG island of OGG1 in lens cortex of ARC. The role of epigenetic change in OGG1 gene in the susceptibility to oxidative stress induced cortical ARC is warranted to further study.

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings.

PubMed Central

Quaite, F. E.; Takayanagi, S.; Ruffini, J.; Sutherland, J. C.; Sutherland, B. M.

1994-01-01

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage. PMID:12244228

Coupling between nucleotide excision repair and gene expression.

PubMed

Cambindo Botto, Adrián E; Muñoz, Juan C; Muñoz, Manuel J

2018-05-17

Gene expression and DNA repair are fundamental processes for life. During the last decade, accumulating experimental evidence point towards different modes of coupling between these processes. Here we discuss the molecular mechanisms by which RNAPII-dependent transcription affects repair by the Nucleotide Excision Repair system (NER) and how NER activity, through the generation of single stranded DNA intermediates and activation of the DNA damage response kinase ATR, drives gene expression in a genotoxic scenario. Since NER-dependent repair is compromised in Xeroderma Pigmentosum (XP) patients, and having in mind that these patients present a high degree of clinical heterogeneity, we speculate that some of the clinical features of XP patients can be explained by misregulation of gene expression.

DNA Repair in Drosophila: Mutagens, Models, and Missing Genes

PubMed Central

Sekelsky, Jeff

2017-01-01

The numerous processes that damage DNA are counterbalanced by a complex network of repair pathways that, collectively, can mend diverse types of damage. Insights into these pathways have come from studies in many different organisms, including Drosophila melanogaster. Indeed, the first ideas about chromosome and gene repair grew out of Drosophila research on the properties of mutations produced by ionizing radiation and mustard gas. Numerous methods have been developed to take advantage of Drosophila genetic tools to elucidate repair processes in whole animals, organs, tissues, and cells. These studies have led to the discovery of key DNA repair pathways, including synthesis-dependent strand annealing, and DNA polymerase theta-mediated end joining. Drosophila appear to utilize other major repair pathways as well, such as base excision repair, nucleotide excision repair, mismatch repair, and interstrand crosslink repair. In a surprising number of cases, however, DNA repair genes whose products play important roles in these pathways in other organisms are missing from the Drosophila genome, raising interesting questions for continued investigations. PMID:28154196

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

PubMed

García-Gómez, Candela; Parages, María L; Jiménez, Carlos; Palma, Armando; Mata, M Teresa; Segovia, María

2012-09-01

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

UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1)*

PubMed Central

Kemp, Michael G.; Lindsey-Boltz, Laura A.; Sancar, Aziz

2015-01-01

The mechanism by which ultraviolet (UV) wavelengths of sunlight trigger or exacerbate the symptoms of the autoimmune disorder lupus erythematosus is not known but may involve a role for the innate immune system. Here we show that UV radiation potentiates STING (stimulator of interferon genes)-dependent activation of the immune signaling transcription factor interferon regulatory factor 3 (IRF3) in response to cytosolic DNA and cyclic dinucleotides in keratinocytes and other human cells. Furthermore, we find that modulation of this innate immune response also occurs with UV-mimetic chemical carcinogens and in a manner that is independent of DNA repair and several DNA damage and cell stress response signaling pathways. Rather, we find that the stimulation of STING-dependent IRF3 activation by UV is due to apoptotic signaling-dependent disruption of ULK1 (Unc51-like kinase 1), a pro-autophagic protein that negatively regulates STING. Thus, deregulation of ULK1 signaling by UV-induced DNA damage may contribute to the negative effects of sunlight UV exposure in patients with autoimmune disorders. PMID:25792739

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

PubMed

Siede, W; Friedberg, A S; Friedberg, E C

1993-09-01

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

Effect of tif expression, irradiation of recipient and presence of plasmid pKM101 on recovery of a marker from a donor exposed to ultraviolet light prior to conjugation.

PubMed

von Wright, A; Bridges, B A

1980-08-01

To detect the effect of the postulated inducible error-prone repair system ('SOS repair') on the bacterial chromosome, an Hfr Escherichia coli strain JC5088 recA was u.v.-irradiated immediately before mating it with recipients in which SOS repair was supposed to be functioning through tif expression, u.v. irradiation or the presence of plasmid pKM101. The recombinant yields of these crosses were compared with those obtained in corresponding crosses with recipients in which SOS repair either was not induced or was totally eliminated by the lexA mutation. No difference in marker recovery efficiency could be detected between these two sets of recipients and thus no induced repair process acting on donor DNA could be demonstrated. The possible reasons for this finding are discussed.

Femtopulse laser-based mask repair in the DUV wavelength regime

NASA Astrophysics Data System (ADS)

Ghadiali, Firoz; Tolani, Vikram; Nagpal, Rajesh; Robinson, Tod; LeClaire, Jeff; Bozak, Ron; Lee, David A.; White, Roy

2006-05-01

Deep ultraviolet (DUV) femtosecond-pulsed laser ablation has numerous highly desirable properties for subtractive photomask defect repair. These qualities include high removal rates, resolution better than the focused spot size, minimized redeposition of the ablated material (rollup and splatter), and a negligible heat affected zone. The optical properties of the photomask result in a broad repair process window because the absorber film (whether Cr or MoSi) and the transmissive substrate allow for a high degree of material removal selectivity. Repair results and process parameters from such a system are examined in light of theoretical considerations. In addition, the practical aspects of the operation of this system in a production mask house environment are reviewed from the standpoint of repair quality, capability, availability, and throughput. Focus is given to the benefit received by the mask shop, and to the technical performance of the system.

Resistance of the Extreme Halophile Halobacterium sp. NRC-1 to Multiple Stresses

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

Gygli, Patrick E.; Prajapati, Surendra; DeVeaux, Linda C.

2009-03-10

The model Archaeon Halobacterium sp. NRC-1 is an extreme halophile known for its resistance to multiple stressors, including electron-beam and ultraviolet radiation. It is a well-developed system with a completely sequenced genome and extensive post-genomic tools for the study of a variety of biological processes. To further understand the mechanisms of Halobacterium's, radiation resistance, we previously reported the selection for multiple independent highly resistant mutants using repeated exposure to high doses of 18-20 MeV electrons using a medical S-band Linac. Molecular analysis of the transcriptional profile of several of these mutants revealed a single common change: upregulation of the rfa3more » operon. These genes encode proteins homologous to the subunits of eukaryotic Replication Protein A (RPA), a DNA binding protein with major roles in DNA replication, recombination, and repair. This operon has also been implicated in a somewhat lesser role in resistance of wild type Halobacterium to ultraviolet radiation, suggesting common mechanisms for resistance. To further understand the mechanism of radiation resistance in the mutant strains, we measured the survival after exposure to both electron-beam and ultraviolet radiation, UV-A, B, and C All mutant strains showed increased resistance to electrons when compared with the parent. However, the mutant strains do not display increased UV resistance, and in one case is more sensitive than the parent strain. Thus, the protective role of increased RPA expression within a cell may be specific to the DNA damage caused by the different physical effects induced by high energy electron-beam radiation.« less

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

NASA Technical Reports Server (NTRS)

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

2009-01-01

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

Day and night variations in the repair of ionizing-radiation-induced DNA damage in mouse splenocytes.

PubMed

Palombo, Philipp; Moreno-Villanueva, Maria; Mangerich, Aswin

2015-04-01

In mammals, biological rhythms synchronize physiological and behavioral processes to the 24-h light-dark (LD) cycle. At the molecular level, self-sustaining processes, such as oscillations of transcription-translation feedback loops, control the circadian clock, which in turn regulates a wide variety of cellular processes, including gene expression and cell cycle progression. Furthermore, previous studies reported circadian oscillations in the repair capacity of DNA lesions specifically repaired by nucleotide excision repair (NER). However, it is so far only poorly understood if DNA repair pathways other than NER are under circadian control, in particular base excision and DNA strand break repair. In the present study, we analyzed potential day and night variations in the repair of DNA lesions induced by ionizing radiation (i.e., mainly oxidative damage and DNA strand breaks) in living mouse splenocytes using a modified protocol of the automated FADU assay. Our results reveal that splenocytes isolated from mice during the light phase (ZT06) displayed higher DNA repair activity than those of the dark phase (ZT18). As analyzed by highly sensitive and accurate qPCR arrays, these alterations were accompanied by significant differences in expression profiles of genes involved in the circadian clock and DNA repair. Notably, the majority of the DNA repair genes were expressed at higher levels during the light phase (ZT06). This included genes of all major DNA repair pathways with the strongest differences observed for genes of base excision and DNA double strand break repair. In conclusion, here we provide novel evidence that mouse splenocytes exhibit significant differences in the repair of IR-induced DNA damage during the LD cycle, both on a functional and on a gene expression level. It will be interesting to test if these findings could be exploited for therapeutic purposes, e.g. time-of-the-day-specific application of DNA-damaging treatments used against blood malignancies. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation and characterization of ultraviolet light-sensitive mutants of the blue-green alga Anacystis nidulans.

NASA Technical Reports Server (NTRS)

Asato, Y.

1972-01-01

Three independently isolated ultraviolet light sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 showed the highest sensitivity to UV by its greatly reduced photoreactivation capacity following irradiation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, thus indicating the presence of excision enzymes involved in dark repair. Under 'black' and 'white' illumination, strain uvs-1 shows photorecovery properties comparable with wild-type cultures. Results indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.

Beyond xeroderma pigmentosum: DNA damage and repair in an ecological context. A tribute to James E. Cleaver.

PubMed

Karentz, Deneb

2015-01-01

The ability to repair DNA is a ubiquitous characteristic of life on Earth and all organisms possess similar mechanisms for dealing with DNA damage, an indication of a very early evolutionary origin for repair processes. James E. Cleaver's career (initiated in the early 1960s) has been devoted to the study of mammalian ultraviolet radiation (UVR) photobiology, specifically the molecular genetics of xeroderma pigmentosum and other human diseases caused by defects in DNA damage recognition and repair. This work by Jim and others has influenced the study of DNA damage and repair in a variety of taxa. Today, the field of DNA repair is enhancing our understanding of not only how to treat and prevent human disease, but is providing insights on the evolutionary history of life on Earth and how natural populations are coping with UVR-induced DNA damage from anthropogenic changes in the environment such as ozone depletion. © 2014 The American Society of Photobiology.

DNA Repair Mechanism Gene, XRCC1A ( Arg194Trp) but not XRCC3 ( Thr241Met) Polymorphism Increased the Risk of Breast Cancer in Premenopausal Females: A Case-Control Study in Northeastern Region of India.

PubMed

Devi, K Rekha; Ahmed, Jishan; Narain, Kanwar; Mukherjee, Kaustab; Majumdar, Gautam; Chenkual, Saia; Zonunmawia, Jason C

2017-12-01

X-ray repair cross complementary group gene is one of the most studied candidate gene involved in different types of cancers. Studies have shown that X-ray repair cross complementary genes are significantly associated with increased risk of breast cancer in females. Moreover, studies have revealed that X-ray repair cross complementary gene polymorphism significantly varies between and within different ethnic groups globally. The present case-control study was aimed to investigate the association of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) polymorphism with the risk of breast cancer in females from northeastern region of India. The present case-control study includes histopathologically confirmed and newly diagnosed 464 cases with breast cancer and 534 apparently healthy neighborhood community controls. Information on sociodemographic factors and putative risk factors were collected from each study participant by conducting face-to-face interviews. Genotyping of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) was carried out by polymerase chain reaction-restriction fragment length polymorphism. For statistical analysis, both univariate and multivariate logistic regression analyses were performed. We also performed stratified analysis to find out the association of X-ray repair cross complementary genes with the risk of breast cancer stratified based on menstrual status. This study revealed that tryptophan allele (R/W-W/W genotype) in X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer (adjusted odds ratio = 1.44, 95% confidence interval = 1.06-1.97, P < .05 for R/W-W/W genotype). Moreover, it was found that tryptophan allele (W/W genotype) at codon 194 of X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer in premenopausal females (crude odds ratio = 1.66, 95% confidence interval = 1.11-2.46, P < .05 for R/W-W/W genotype). The present study did not reveal any significant association of X-ray repair cross complementary 3 (Thr241Met) polymorphism with the risk of breast cancer. The present study has explored that X-ray repair cross complementary 1A (Arg194Trp) gene polymorphism is significantly associated with the increased risk of breast cancer in premenopausal females from northeastern region of India which may be beneficial for prognostic purposes.

DNA Repair Mechanism Gene, XRCC1A (Arg194Trp) but not XRCC3 (Thr241Met) Polymorphism Increased the Risk of Breast Cancer in Premenopausal Females: A Case–Control Study in Northeastern Region of India

PubMed Central

Ahmed, Jishan; Narain, Kanwar; Mukherjee, Kaustab; Majumdar, Gautam; Chenkual, Saia; Zonunmawia, Jason C.

2017-01-01

X-ray repair cross complementary group gene is one of the most studied candidate gene involved in different types of cancers. Studies have shown that X-ray repair cross complementary genes are significantly associated with increased risk of breast cancer in females. Moreover, studies have revealed that X-ray repair cross complementary gene polymorphism significantly varies between and within different ethnic groups globally. The present case–control study was aimed to investigate the association of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) polymorphism with the risk of breast cancer in females from northeastern region of India. The present case–control study includes histopathologically confirmed and newly diagnosed 464 cases with breast cancer and 534 apparently healthy neighborhood community controls. Information on sociodemographic factors and putative risk factors were collected from each study participant by conducting face-to-face interviews. Genotyping of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) was carried out by polymerase chain reaction-restriction fragment length polymorphism. For statistical analysis, both univariate and multivariate logistic regression analyses were performed. We also performed stratified analysis to find out the association of X-ray repair cross complementary genes with the risk of breast cancer stratified based on menstrual status. This study revealed that tryptophan allele (R/W-W/W genotype) in X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer (adjusted odds ratio = 1.44, 95% confidence interval = 1.06-1.97, P < .05 for R/W-W/W genotype). Moreover, it was found that tryptophan allele (W/W genotype) at codon 194 of X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer in premenopausal females (crude odds ratio = 1.66, 95% confidence interval = 1.11-2.46, P < .05 for R/W-W/W genotype). The present study did not reveal any significant association of X-ray repair cross complementary 3 (Thr241Met) polymorphism with the risk of breast cancer. The present study has explored that X-ray repair cross complementary 1A (Arg194Trp) gene polymorphism is significantly associated with the increased risk of breast cancer in premenopausal females from northeastern region of India which may be beneficial for prognostic purposes. PMID:29332455

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

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

Flejter, W.L.; McDaniel, L.D.; Johns, D.

1992-01-01

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2)more » gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.« less

DR-GAS: a database of functional genetic variants and their phosphorylation states in human DNA repair systems.

PubMed

Sehgal, Manika; Singh, Tiratha Raj

2014-04-01

We present DR-GAS(1), a unique, consolidated and comprehensive DNA repair genetic association studies database of human DNA repair system. It presents information on repair genes, assorted mechanisms of DNA repair, linkage disequilibrium, haplotype blocks, nsSNPs, phosphorylation sites, associated diseases, and pathways involved in repair systems. DNA repair is an intricate process which plays an essential role in maintaining the integrity of the genome by eradicating the damaging effect of internal and external changes in the genome. Hence, it is crucial to extensively understand the intact process of DNA repair, genes involved, non-synonymous SNPs which perhaps affect the function, phosphorylated residues and other related genetic parameters. All the corresponding entries for DNA repair genes, such as proteins, OMIM IDs, literature references and pathways are cross-referenced to their respective primary databases. DNA repair genes and their associated parameters are either represented in tabular or in graphical form through images elucidated by computational and statistical analyses. It is believed that the database will assist molecular biologists, biotechnologists, therapeutic developers and other scientific community to encounter biologically meaningful information, and meticulous contribution of genetic level information towards treacherous diseases in human DNA repair systems. DR-GAS is freely available for academic and research purposes at: http://www.bioinfoindia.org/drgas. Copyright © 2014 Elsevier B.V. All rights reserved.

Reactive oxygen-mediated damage to a human DNA replication and repair protein.

PubMed

Montaner, Beatriz; O'Donovan, Peter; Reelfs, Olivier; Perrett, Conal M; Zhang, Xiaohong; Xu, Yao-Zhong; Ren, Xiaolin; Macpherson, Peter; Frith, David; Karran, Peter

2007-11-01

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

Mutagenic effect of accelerated heavy ions on bacterial cells

NASA Astrophysics Data System (ADS)

Boreyko, A. V.; Krasavin, E. A.

2011-11-01

The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value ( L max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L max is ≈ 100 and 50 keV/μm, respectively. The differences in the L max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.

Epigenetic changes of DNA repair genes in cancer.

PubMed

Lahtz, Christoph; Pfeifer, Gerd P

2011-02-01

'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast.

PubMed

Saini, Natalie; Roberts, Steven A; Sterling, Joan F; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

2017-05-01

Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis. Published by Elsevier B.V.

Adeno-associated virus inverted terminal repeats stimulate gene editing.

PubMed

Hirsch, M L

2015-02-01

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

Mutagen Sensitivity and DNA Repair Gene Polymorphisms in Hereditary and Sporadic Breast Cancer

DTIC Science & Technology

2005-03-01

exposures trigger breast cancer in the general population; BRCA] mutation carriers are just substantially more susceptible to these gene -environment...repair complex Genes that affect BRCAJ mutation penetrance might be those involved in DNA repair, because BRCA1 is itself believed to be involved in...without founder mutations (187delAG and 5382insC) had complete BRCA] and BRCA2 gene sequencing. Some patients are related (n=34). Patient demographics

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Repair-dependent cell radiation survival and transformation: an integrated theory.

PubMed

Sutherland, John C

2014-09-07

The repair-dependent model of cell radiation survival is extended to include radiation-induced transformations. The probability of transformation is presumed to scale with the number of potentially lethal damages that are repaired in a surviving cell or the interactions of such damages. The theory predicts that at doses corresponding to high survival, the transformation frequency is the sum of simple polynomial functions of dose; linear, quadratic, etc, essentially as described in widely used linear-quadratic expressions. At high doses, corresponding to low survival, the ratio of transformed to surviving cells asymptotically approaches an upper limit. The low dose fundamental- and high dose plateau domains are separated by a downwardly concave transition region. Published transformation data for mammalian cells show the high-dose plateaus predicted by the repair-dependent model for both ultraviolet and ionizing radiation. For the neoplastic transformation experiments that were analyzed, the data can be fit with only the repair-dependent quadratic function. At low doses, the transformation frequency is strictly quadratic, but becomes sigmodial over a wider range of doses. Inclusion of data from the transition region in a traditional linear-quadratic analysis of neoplastic transformation frequency data can exaggerate the magnitude of, or create the appearance of, a linear component. Quantitative analysis of survival and transformation data shows good agreement for ultraviolet radiation; the shapes of the transformation components can be predicted from survival data. For ionizing radiations, both neutrons and x-rays, survival data overestimate the transforming ability for low to moderate doses. The presumed cause of this difference is that, unlike UV photons, a single x-ray or neutron may generate more than one lethal damage in a cell, so the distribution of such damages in the population is not accurately described by Poisson statistics. However, the complete sigmodial dose-response data for neoplastic transformations can be fit using the repair-dependent functions with all parameters determined only from transformation frequency data.

Mutation of MSH3 in endometrial cancer and evidence for its functional role in heteroduplex repair.

PubMed

Risinger, J I; Umar, A; Boyd, J; Berchuck, A; Kunkel, T A; Barrett, J C

1996-09-01

Many human tumours have length alterations in repetitive sequence elements. Although this microsatellite instability has been attributed to mutations in four DNA mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds, many sporadic tumours exhibit instability but no detectable mutations in these genes. It is therefore of interest to identify other genes that contribute to this instability. In yeast, mutations in several genes, including RTH and MSH3, cause microsatellite instability. Thus, we screened 16 endometrial carcinomas with microsatellite instability for alterations in FEN1 (the human homolog of RTH) and in MSH3 (refs 12-14). Although we found no FEN1 mutations, a frameshift mutation in MSH3 was observed in an endometrial carcinoma and in an endometrial carcinoma cell line. Extracts of the cell line were deficient in repair of DNA substrates containing mismatches or extra nucleotides. Introducing chromosome 5, encoding the MSH3 gene, into the mutant cell line increased the stability of some but not all microsatellites. Extracts of these cells repaired certain substrates containing extra nucleotides, but were deficient in repair of those containing mismatches or other extra nucleotides. A subsequent search revealed a second gene mutation in HHUA cells, a missense mutation in the MSH6 gene. Together the data suggest that the MSH3 gene encodes a product that functions in repair of some but not all pre-mutational intermediates, its mutation in tumours can result in genomic instability and, as in yeast, MSH3 and MSH6 are partially redundant for mismatch repair.

Concepts in Gene Therapy for Cartilage Repair

PubMed Central

Steinert, Andre F.; Nöth, Ulrich; Tuan, Rocky S.

2009-01-01

Summary Once articular cartilage is injured, it has a very limited capacity for self-repair. Although current surgical therapeutic procedures to cartilage repair are clinically useful, they cannot restore a normal articular surface. Current research offers a growing number of bioactive reagents, including proteins and nucleic acids, that may be used to augment different aspects of the repair process. As these agents are difficult to administer effectively, gene transfer approaches are being developed to provide their sustained synthesis at sites of repair. To augment regeneration of articular cartilage, therapeutic genes can be delivered to the synovium, or directly to the cartilage lesion. Gene delivery to the cells of the synovial lining is generally considered more suitable for chondroprotective approaches, based on the expression of anti-inflammatory mediators. Gene transfer targeted to cartilage defects can be achieved by either direct vector administration to cells located at or surrounding the defects, or by transplantation of genetically modified chondrogenic cells into the defect. Several studies have shown that exogenous cDNAs encoding growth factors can be delivered locally to sites of cartilage damage, where they are expressed at therapeutically relevant levels. Furthermore, data is beginning to emerge indicating, that efficient delivery and expression of these genes is capable of influencing a repair response toward the synthesis of a more hyaline cartilage repair tissue in vivo. This review presents the current status of gene therapy for cartilage healing and highlights some of the remaining challenges. PMID:18313477

Genomic footprints of dryland stress adaptation in Egyptian fat-tail sheep and their divergence from East African and western Asia cohorts.

PubMed

Mwacharo, Joram M; Kim, Eui-Soo; Elbeltagy, Ahmed R; Aboul-Naga, Adel M; Rischkowsky, Barbara A; Rothschild, Max F

2017-12-15

African indigenous sheep are classified as fat-tail, thin-tail and fat-rump hair sheep. The fat-tail are well adapted to dryland environments, but little is known on their genome profiles. We analyzed patterns of genomic variation by genotyping, with the Ovine SNP50K microarray, 394 individuals from five populations of fat-tail sheep from a desert environment in Egypt. Comparative inferences with other East African and western Asia fat-tail and European sheep, reveal at least two phylogeographically distinct genepools of fat-tail sheep in Africa that differ from the European genepool, suggesting separate evolutionary and breeding history. We identified 24 candidate selection sweep regions, spanning 172 potentially novel and known genes, which are enriched with genes underpinning dryland adaptation physiology. In particular, we found selection sweeps spanning genes and/or pathways associated with metabolism; response to stress, ultraviolet radiation, oxidative stress and DNA damage repair; activation of immune response; regulation of reproduction, organ function and development, body size and morphology, skin and hair pigmentation, and keratinization. Our findings provide insights on the complexity of genome architecture regarding dryland stress adaptation in the fat-tail sheep and showcase the indigenous stocks as appropriate genotypes for adaptation planning to sustain livestock production and human livelihoods, under future climates.

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.

PubMed

Pritchard, Colin C; Mateo, Joaquin; Walsh, Michael F; De Sarkar, Navonil; Abida, Wassim; Beltran, Himisha; Garofalo, Andrea; Gulati, Roman; Carreira, Suzanne; Eeles, Rosalind; Elemento, Olivier; Rubin, Mark A; Robinson, Dan; Lonigro, Robert; Hussain, Maha; Chinnaiyan, Arul; Vinson, Jake; Filipenko, Julie; Garraway, Levi; Taplin, Mary-Ellen; AlDubayan, Saud; Han, G Celine; Beightol, Mallory; Morrissey, Colm; Nghiem, Belinda; Cheng, Heather H; Montgomery, Bruce; Walsh, Tom; Casadei, Silvia; Berger, Michael; Zhang, Liying; Zehir, Ahmet; Vijai, Joseph; Scher, Howard I; Sawyers, Charles; Schultz, Nikolaus; Kantoff, Philip W; Solit, David; Robson, Mark; Van Allen, Eliezer M; Offit, Kenneth; de Bono, Johann; Nelson, Peter S

2016-08-04

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites.

PubMed Central

Lieb, M

1987-01-01

Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations. The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences. Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system. In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable. VSP mismatch repair was greatly reduced in a dcm Escherichia coli mutant, in which 5-methylcytosine was not methylated. However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm+ bacteria. These results indicate that the product of gene dcm has a repair function in addition to its methylase activity. PMID:2959653

Mechanisms of radiation-induced gene responses

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

Woloschak, G.E.; Paunesku, T.

1996-10-01

In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts;more » however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.« less

Gene repair of an Usher syndrome causing mutation by zinc-finger nuclease mediated homologous recombination.

PubMed

Overlack, Nora; Goldmann, Tobias; Wolfrum, Uwe; Nagel-Wolfrum, Kerstin

2012-06-26

Human Usher syndrome (USH) is the most frequent cause of inherited deaf-blindness. It is clinically and genetically heterogeneous, assigned to three clinical types of which the most severe type is USH1. No effective treatment for the ophthalmic component of USH exists. Gene augmentation is an attractive strategy for hereditary retinal diseases. However, several USH genes, like USH1C, are expressed in various isoforms, hampering gene augmentation. As an alternative treatment strategy, we applied the zinc-finger nuclease (ZFN) technology for targeted gene repair of an USH1C, causing mutation by homologous recombination. We designed ZFNs customized for the p.R31X nonsense mutation in Ush1c. We evaluated ZFNs for DNA cleavage capability and analyzed ZFNs biocompatibilities by XTT assays. We demonstrated ZFNs mediated gene repair on genomic level by digestion assays and DNA sequencing, and on protein level by indirect immunofluorescence and Western blot analyses. The specifically designed ZFNs did not show cytotoxic effects in a p.R31X cell line. We demonstrated that ZFN induced cleavage of their target sequence. We showed that simultaneous application of ZFN and rescue DNA induced gene repair of the disease-causing mutation on the genomic level, resulting in recovery of protein expression. In our present study, we analyzed for the first time ZFN-activated gene repair of an USH gene. The data highlight the ability of ZFNs to induce targeted homologous recombination and mediate gene repair in USH. We provide further evidence that the ZFN technology holds great potential to recover disease-causing mutations in inherited retinal disorders.

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

PubMed

Gruszka, Damian; Marzec, Marek; Szarejko, Iwona

2012-06-14

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

Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

NASA Astrophysics Data System (ADS)

Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

2016-07-01

Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

Sequence homology and expression profile of genes associated with DNA repair pathways in Mycobacterium leprae.

PubMed

Sharma, Mukul; Vedithi, Sundeep Chaitanya; Das, Madhusmita; Roy, Anindya; Ebenezer, Mannam

2017-01-01

Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae. T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%), 11 hypothetical proteins (18%), and 14 pseudogenes (23%). All these genes have homologs in M. tuberculosis and 49 (80.32%) in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps. It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA). The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes) were analyzed using quantitative Polymerase Chain Reaction (qPCR) assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the direct repair pathway. This study provided preliminary information on the potential DNA repair pathways that are extant in M. leprae and the associated genes.

Cancer prevention, the need to preserve the integrity of the genome at all cost.

PubMed

Okafor, M T; Nwagha, T U; Anusiem, C; Okoli, U A; Nubila, N I; Al-Alloosh, F; Udenyia, I J

2018-05-01

The entire genetic information carried by an organism makes up its genome. Genes have a diverse number of functions. They code different proteins for normal proliferation of cells. However, changes in the base sequence of genes affect their protein by-products which act as messengers for normal cellular functions such as proliferation and repairs. Salient processes for maintaining the integrity of the genome are hinged on intricate mechanisms put in place for the evolution to tackle genomic stresses. To discuss how cells sense and repair damage to their deoxyribonucleic acid (DNA) as well as to highlight how defects in the genes involved in DNA repair contribute to cancer development. Methodology: Online searches on the following databases such as Google Scholar, PubMed, Biomed Central, and SciELO were done. Attempt was made to review articles with keywords such as cancer, cell cycle, tumor suppressor genes, and DNA repair. The cell cycle, tumor suppression genes, DNA repair mechanism, as well as their contribution to cancer development, were discussed and reviewed. Knowledge on how cells detect and repair DNA damage through an array of mechanisms should allay our anxiety as regards cancer development. More studies on DNA damage detection and repair processes are important toward a holistic approach to cancer treatment.

Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

PubMed Central

Christensen, Alan C.

2014-01-01

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

Ultraviolet light induction of diphtheria toxin-resistant mutations in normal and DNA repair-deficient human and Chinese hamster fibroblasts

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

Trosko, J.E.; Schultz, R.S.; Chang, C.C.

1980-01-01

The role on unrepaired DNA lesions in the production of mutations is suspected of contributing to the initiation phase of carcinogenesis. Since the molecular basis of mutagenesis is not understood in eukaryotic cells, development of new genetic markers for quantitative in vitro measurement of mutations for mammalian cells is needed. Furthermore, mammalian cells, genetically deficient for various DNA repair enzymes, will be needed to study the role of unrepaired DNA lesions in mutagenesis. The results in this report relate to preliminary attempts to characterize the diphtheria toxin resistance marker as a useful quantitative genetic marker in human cells and tomore » isolate and characterize various DNA repair-deficient Chinese hamster cells.« less

Implication of SUMO E3 ligases in nucleotide excision repair.

PubMed

Tsuge, Maasa; Kaneoka, Hidenori; Masuda, Yusuke; Ito, Hiroki; Miyake, Katsuhide; Iijima, Shinji

2015-08-01

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

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

Gupta, P.K.; Sirover, M.A.

1984-10-01

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior tomore » their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. 62 references, 3 figures, 2 tables.« less

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

PubMed

Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

2018-04-15

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

Reduced expression of DNA repair genes and chemosensitivity in 1p19q codeleted lower-grade gliomas.

PubMed

Tang, Lei; Deng, Lu; Bai, Harrison X; Sun, James; Neale, Natalie; Wu, Jing; Wang, Yinyan; Chang, Ken; Huang, Raymond Y; Zhang, Paul J; Li, Xuejun; Xiao, Bo; Cao, Ya; Tao, Yongguang; Yang, Li

2018-06-19

Lower-grade gliomas (LGGs, defined as WHO grades II and III) with 1p19q codeletion have increased chemosensitivity when compared to LGGs without 1p19q codeletion, but the mechanism is currently unknown. RNAseq data from 515 LGG patients in the Cancer Genome Atlas (TCGA) were analyzed to compare the effect of expression of the 9 DNA repair genes located on chromosome arms 1p and 19q on progression free survival (PFS) and overall survival (OS) between patients who received chemotherapy and those who did not. Chemosensitivity of cells with DNA repair genes knocked down was tested using MTS cell proliferation assay in HS683 cell line and U251 cell line. The expression of 9 DNA repair genes on 1p and 19q was significantly lower in 1p19q-codeleted tumors (n = 175) than in tumors without the codeletion (n = 337) (p < 0.001). In LGG patients who received chemotherapy, lower expression of LIG1, POLD1, PNKP, RAD54L and MUTYH was associated with longer PFS and OS. This difference between chemotherapy and non-chemotherapy groups in the association of gene expression with survival was not observed in non-DNA repair genes located on chromosome arms 1p and 19q. MTS assays showed that knockdown of DNA repair genes LIG1, POLD1, PNKP, RAD54L and MUTYH significantly inhibited recovery in response to temozolomide when compared with control group (p < 0.001). Our results suggest that reduced expression of DNA repair genes on chromosome arms 1p and 19q may account for the increased chemosensitivity of LGGs with 1p19q codeletion.

[Polymorphism of genes encoding proteins of DNA repair vs. occupational and environmental exposure to lead, arsenic and pesticides].

PubMed

Bukowski, Karol; Woźniak, Katarzyna

2018-03-09

Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(2):225-235. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

PubMed Central

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

2013-01-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

Precision genome editing in the CRISPR era.

PubMed

Salsman, Jayme; Dellaire, Graham

2017-04-01

With the introduction of precision genome editing using CRISPR-Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR-Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR-Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

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

Venema, J.; van Hoffen, A.; Karcagi, V.

1991-08-01

The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5{prime} part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimersmore » removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3{prime} part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.« less

Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

PubMed Central

Ng, P; Baker, M D

1999-01-01

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929

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

PubMed

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

1998-11-20

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

DNA Damage Response Genes and the Development of Cancer Metastasis

PubMed Central

Broustas, Constantinos G.; Lieberman, Howard B.

2014-01-01

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

MicroRNAs in Skin Response to UV Radiation

PubMed Central

Syed, Deeba N.; Khan, Mohammad Imran; Shabbir, Maria; Mukhtar, Hasan

2014-01-01

Solar ultraviolet (UV) radiation, an ubiquitous environmental carcinogen, is classified depending on the wave-length, into three regions; short-wave UVC (200–280 nm), mid-wave UVB (280–320 nm), and long-wave UVA (320–400 nm). The human skin, constantly exposed to UV radiation, particularly the UVB and UVA components, is vulnerable to its various deleterious effects such as erythema, photoaging, immunosuppression and cancer. To counteract these and for the maintenance of genomic integrity, cells have developed several protective mechanisms including DNA repair, cell-cycle arrest and apoptosis. The network of damage sensors, signal transducers, mediators, and various effector proteins is regulated through changes in gene expression. MicroRNAs (miRNAs), a group of small non-coding RNAs, act as post-transcriptional regulators through binding to complementary sequences in the 3′-untranslated region of their target genes, resulting in either translational repression or target degradation. Recent studies show that miRNAs add an additional layer of complexity to the intricately controlled cellular responses to UV radiation. This review summarizes our current knowledge of the role of miRNAs in the regulation of the human skin response upon exposure to UV radiation. PMID:23834148

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

PubMed

Ager, D D; Radul, J A

1992-12-01

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.

Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

PubMed

Das, Raima; Ghosh, Sankar Kumar

2017-04-01

DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

Total Defense + Repair: A Novel Concept in Solar Protection and Skin Rejuvenation.

PubMed

McDaniel, David H; Hamzavi, Iltefat H; Zeichner, Joshua A; Fabi, Sabrina G; Bucay, Vivian W; Harper, Julie C; Comstock, Jody A; Makino, Elizabeth T; Mehta, Rahul C; Vega, Virginia L

2015-07-01

For more than a century, solar radiation has been known to contribute significantly to the extrinsic aging of skin. Until recently, this was almost exclusively attributed to the photodamage caused by ultraviolet (UV) light. However, a growing body of evidence now indicates that both infrared (IR) and visible light may also contribute to extrinsic skin aging. Infrared radiation, comprised of IR-A, IR-B, and IR-C, accounts for 54.3% of the total solar radiation reaching the skin. Studies have shown that IR radiation is also responsible for skin aging. Thus, IR-A radiation regulates hundreds of genes in skin, with roles in extracellular matrix (ECM) homeostasis regulation, apoptosis, cell growth, and stress responses. IR-B and IR-C radiation are primarily responsible for the increase in skin temperature associated with solar exposure, and are implicated in heat-related skin destruction of collagen and elastin, which is characterized by an increase in the expression of matrix metalloproteinases (MMPs). The contribution of visible light to photoaging is less well understood; however, some preliminary indication associates visible light with the upregulation of MMPs' expression, DNA damage, and keratinocyte proliferation. Interestingly, the common denominator that links skin damage to the different solar wavelengths is the enhanced production of reactive molecule species (RMS) and therewith increased oxidative stress. SkinMedica® Total Defense + Repair (TD+R; SkinMedica Inc., an Allergan company, Irvine, CA) is a "superscreen," which combines broad spectrum UV protection with a unique blend of antioxidants (SOL-IR Advanced Antioxidant Complex™) that provide protection from IR radiation while promoting skin repair. Preclinical studies have indicated that TD+R SPF34 prevents the formation of UV-induced sunburn cells and cyclobutane pyrimidine dimers while preserving or improving the expression of ECM genes. In addition, it prevents IR-A-triggered fragmentation of elastin fibers and expression of MMP-1. Initial clinical studies indicate that TDR+R SPF34 reduces the increase in surface temperature seen with IR radiation. A significant improvement in the appearance of lines and wrinkles was reported as early as week 2 in patients using TDR+R SPF34. In summary, we observed that the unique blend of antioxidants present in TD+R acts in harmony with SPF active ingredients, expanding solar protection beyond UV radiation and counterbalancing the deleterious effects of free radicals on skin cells by promoting endogenous repair.

Genetic variation in a DNA double strand break repair gene in saudi population: a comparative study with worldwide ethnic groups.

PubMed

Areeshi, Mohammed Yahya

2013-01-01

DNA repair capacity is crucial in maintaining cellular functions and homeostasis. However, it can be altered based on DNA sequence variations in DNA repair genes and this may lead to the development of many diseases including malignancies. Identification of genetic polymorphisms responsible for reduced DNA repair capacity is necessary for better prevention. Homologous recombination (HR), a major double strand break repair pathway, plays a critical role in maintaining the genome stability. The present study was performed to determine the frequency of the HR gene XRCC3 Exon 7 (C18067T, rs861539) polymorphisms in Saudi Arabian population in comparison with epidemiological studies by "MEDLINE" search to equate with global populations. The variant allelic (T) frequency of XRCC3 (C>T) was found to be 39%. Our results suggest that frequency of XRCC3 (C>T) DNA repair gene exhibits distinctive patterns compared with the Saudi Arabian population and this might be attributed to ethnic variation. The present findings may help in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

PubMed Central

Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

Orbital amelanotic melanoma in xeroderma pigmentosum: A rare association

PubMed Central

Amitava, Abadan K; Mehdi, Ghazala; Sharma, Rajeev; Alam, Mohammad S

2008-01-01

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder of DNA repair in which the body′s normal ability to repair damage caused by ultraviolet light is deficient. This leads to a 1000-fold increased risk of cutaneous and ocular neoplasms. Ocular neoplasms occurring in XP in order of frequency are squamous cell carcinoma, basal cell carcinoma and melanoma. Malignant melanomas occur at an early age in patients with XP. We report a case of XP with massive orbital melanoma in an eight-year-old boy which is unique due to its amelanotic presentation confirmed histopathologically. PMID:18711275

Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

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

PubMed Central

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

2011-01-01

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

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Xeroderma pigmentosum complementation group F: Report of a case and review of Japanese patients.

PubMed

Tofuku, Yukari; Nobeyama, Yoshimasa; Kamide, Ryoichi; Moriwaki, Shinichi; Nakagawa, Hidemi

2015-09-01

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by extraordinary sensitivity to sunlight, resulting in cutaneous malignant tumors. Among XP, XP-F presents relatively uniquely in Japanese. To clarify the characteristics of this group, we describe a case of XP-F and review Japanese cases previously reported. A 50-year-old Japanese woman was referred to us with multiple, variously sized, light- or dark-brown macules on the face and sunlight-exposed extremities. She had experienced bulla formation with approximately 10 min of sunlight exposure during her elementary school years. Her parents had been first cousins, and her mother and sister had photosensitivity. She showed no neurological or developmental abnormalities. Ultraviolet (UV) irradiation testing revealed normal levels for minimal erythema dose with UV-A and UV-B. Sensitivity to UV-C and DNA repair ability in the patient's fibroblasts were indicated between that in normal individuals and that in an XP-A patient. Complementation assay revealed that transfection of the XPF gene led most efficient DNA repair compared with the other XP genes. Therefore, the patient was diagnosed with XP-F. Twenty-three cases of Japanese patients (six males, 17 females) with XP-F have been reported, including the present case. Our review suggested a relatively high prevalence of 50% (11/22) for cutaneous malignant tumors. A significant difference was evident in the mean age at first medical consultation between patients with cutaneous malignant tumors (53.6 years) and patients without such tumors (30.8 years). This suggests that cutaneous malignant tumors could occur in the age range of 30-50 years in XP-F patients. © 2015 Japanese Dermatological Association.

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

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

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age, massive programmed cell death, and the release of transcriptional gene silencing. Our data suggests that plants can initiate programmed cell death to eliminate damaged cells despite the absence of p53 in plant genome.« less

The Role of Inducible DNA Repair in W-Reactivation and Related Phenomena.

DTIC Science & Technology

1981-10-14

unexcised dimers in X DNA. This was consistent with the finding of Tomilin and Mosevitskaya (1975) which showed that the UV-endonuclease from Micrococcus ...of DNA in vitro with UV-endonuclease from Micrococcus luteus. Mutat. Res. 27, 147-156 (1975) Tomizawa, J., Ogawa, T.: Effect of ultraviolet irradiation

Effect of smokeless tobacco and tobacco-related chemical carcinogens on survival of ultraviolet light-inactivated herpes simplex virus

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

Dokko, H.; Min, P.S.; Cherrick, H.M.

1991-04-01

Low doses of ultraviolet (UV) light, x-rays, photodynamic treatment, or aflatoxins increase the survival of UV-irradiated virus in cells. This effect is postulated to occur by enhancement of the error-prone cellular repair function, which could also be associated with oncogenic cell transformation. The present study was designed to investigate whether treatment of green monkey kidney cells with water extract of snuff (snuff extract), benzo(a)pyrene, nicotine, or tobacco-specific N'-nitrosamines would result in enhanced survival of UV-irradiated herpes simplex virus (HSV). Exposure of the cells with snuff extract, benzo(a)pyrene, N'-nitrosonornicotine, or 4-(N-methyl-N'-nitrosamino)-1-(3-pyridyl)-1-butanone resulted in an enhancement of survival of UV-irradiated HSV typemore » 1 compared with the control whereas exposure of the cells with nicotine did not. These data indicate that the water-extractable component of snuff and tobacco-related chemical carcinogens increase the cellular repair mechanism and provides for increased survival of UV-irradiated HSV.« less

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

PubMed

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

2003-04-10

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic. Copyright 2003 Wiley-Liss, Inc.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms.

PubMed

Azevedo, Ana P; Silva, Susana N; De Lima, João P; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-06-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog ( E. coli ) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms

PubMed Central

Azevedo, Ana P.; Silva, Susana N.; De Lima, João P.; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-01-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility. PMID:28599464

Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis

PubMed Central

Capilla, Amalia; Karachentsev, Dmitry; Patterson, Rachel A.; Hermann, Anita; Juarez, Michelle T.; McGinnis, William

2017-01-01

The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). In this paper we explore the involvement of the Toll/NF-κB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spätzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spätzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal–ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci. PMID:28289197

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Final progress report

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

Hawley, R.S.

1998-11-01

This research focuses on the structure of the mei-41 gene and elucidation of the role the mei-41 gene product plays in both recombination and repair. Genetic and molecular studies are continuing on the mus308 locus and the mus312 and mei-9 genes. The author views mus312 as a very likely candidate for a gene required for both chromosome pairing/synopsis and for double strand break repair. A thorough genetic study has been initiated of this locus and of the cytology of the meiotic and mitotic defects of mutations at this locus.

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

PubMed Central

Jiménez, Diego Javier; Andreote, Fernando Dini; Chaves, Diego; Montaña, José Salvador; Osorio-Forero, Cesar; Junca, Howard; Zambrano, María Mercedes; Baena, Sandra

2012-01-01

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. PMID:23251687

p53 downregulates the Fanconi anaemia DNA repair pathway

PubMed Central

Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

2016-01-01

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop. PMID:27033104

p53 downregulates the Fanconi anaemia DNA repair pathway.

PubMed

Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

2016-04-01

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

Dissecting DNA repair in adult high grade gliomas for patient stratification in the post-genomic era

PubMed Central

Perry, Christina; Agarwal, Devika; Abdel-Fatah, Tarek M.A.; Lourdusamy, Anbarasu; Grundy, Richard; Auer, Dorothee T.; Walker, David; Lakhani, Ravi; Scott, Ian S.; Chan, Stephen; Ball, Graham; Madhusudan, Srinivasan

2014-01-01

Deregulation of multiple DNA repair pathways may contribute to aggressive biology and therapy resistance in gliomas. We evaluated transcript levels of 157 genes involved in DNA repair in an adult glioblastoma Test set (n=191) and validated in ‘The Cancer Genome Atlas’ (TCGA) cohort (n=508). A DNA repair prognostic index model was generated. Artificial neural network analysis (ANN) was conducted to investigate global gene interactions. Protein expression by immunohistochemistry was conducted in 61 tumours. A fourteen DNA repair gene expression panel was associated with poor survival in Test and TCGA cohorts. A Cox multivariate model revealed APE1, NBN, PMS2, MGMT and PTEN as independently associated with poor prognosis. A DNA repair prognostic index incorporating APE1, NBN, PMS2, MGMT and PTEN stratified patients in to three prognostic sub-groups with worsening survival. APE1, NBN, PMS2, MGMT and PTEN also have predictive significance in patients who received chemotherapy and/or radiotherapy. ANN analysis of APE1, NBN, PMS2, MGMT and PTEN revealed interactions with genes involved in transcription, hypoxia and metabolic regulation. At the protein level, low APE1 and low PTEN remain associated with poor prognosis. In conclusion, multiple DNA repair pathways operate to influence biology and clinical outcomes in adult high grade gliomas. PMID:25026297

The Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula

PubMed Central

Pagano, Andrea; Araújo, Susana de Sousa; Macovei, Anca; Leonetti, Paola; Balestrazzi, Alma

2017-01-01

This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA) during Medicago truncatula seed germination, with emphasis on the seed repair response. Seeds treated with H2O and TSA (10 and 20 μM) were collected during imbibition (8 h) and at the radicle protrusion phase. Biometric data showed delayed germination and impaired seedling growth in TSA-treated samples. Comet assay, performed on radicles at the protrusion phase and 4-days old M. truncatula seedlings, revealed accumulation of DNA strand breaks upon exposure to TSA. Activation of DNA repair toward TSA-mediated genotoxic damage was evidenced by the up-regulation of MtOGG1(8-OXOGUANINE GLYCOSYLASE/LYASE) gene involved in the removal of oxidative DNA lesions, MtLIGIV(LIGASE IV) gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and TDP(TYROSYL-DNA PHOSPHODIESTERASE) genes encoding the multipurpose DNA repair enzymes tyrosyl-DNA phosphodiesterases. Since radical scavenging can prevent DNA damage, the specific antioxidant activity (SAA) was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu reagent assays. Fluctuations of SAA were observed in TSA-treated seeds/seedlings concomitant with the up-regulation of antioxidant genes MtSOD(SUPEROXIDE DISMUTASE, MtAPX(ASCORBATE PEROXIDASE) and MtMT2(TYPE 2 METALLOTHIONEIN). Chromatin remodeling, required to facilitate the access of DNA repair enzymes at the damaged sites, is also part of the multifaceted seed repair response. To address this aspect, still poorly explored in plants, the MtTRRAP(TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN) gene was analyzed. TRRAP is a transcriptional adaptor, so far characterized only in human cells where it is needed for the recruitment of histone acetyltransferase complexes to chromatin during DNA repair. The MtTRRAP gene and the predicted interacting partners MtHAM2 (HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY) and MtADA2A (TRANSCRIPTIONAL ADAPTOR) showed tissue- and dose-dependent fluctuations in transcript levels. PCA (Principal Component Analysis) and correlation analyses suggest for a new putative link between DNA repair and chromatin remodeling that involves MtOGG1 and MtTRRAP genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers. PMID:29184569

Mutation and repair in an ultraviolet-sensitive Chinese hamster ovary cell line

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

Wood, R.D.

1981-11-01

An ultraviolet (UV) light-sensitive mutant of Chinese hamster ovary cells (CHO) has been isolated and characterized with respect to a number of post-irradiation responses. The UV-sensitive mutant, termed 43-3B, has the same growth rate and chromosome number as the wild-type CHO-9. 43-3B is hypersensitive to the lethal effects of UV light (D/sub 0/ of 0.3 J/m/sup 2/ as compared to 3.2 J/m/sup 2/ for the wild-type). A marked UV-hypermutability is observed in 43-3B as compared to the wild-type, as measured with markers for induced resistance to 6-thioguanine, ouabain, and diphtheria toxin. A factor of 38 to 65 more mutations aremore » induced per unit fluence in 43-3B than in CHO-9. The UV-sensitive mutant is also sensitive to killing by simulated solar light, although the D/sub 0/ ratio is not as great as for germicidal UV. 43-3B exhibits only about 17% of the wild-type level of UV-stimulated DNA repair synthesis, as measured by autoradiography of G/sub 1/ phase cells. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild-type CHO-9, but little recovery was seen in 43-3B. The present investigation demonstrates that a sensitive/wild-type pair of CHO cell lines can be used in comparative studies to determine the involvement of repair in a wide range of post-irradiation phenomena.« less

Metagenomic insights into ultraviolet disinfection effects on antibiotic resistome in biologically treated wastewater.

PubMed

Hu, Qing; Zhang, Xu-Xiang; Jia, Shuyu; Huang, Kailong; Tang, Junying; Shi, Peng; Ye, Lin; Ren, Hongqiang

2016-09-15

High-throughput sequencing-based metagenomic approaches were used to comprehensively investigate ultraviolet effects on the microbial community structure, and diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in biologically treated wastewater. After ultraviolet radiation, some dominant genera, like Aeromonas and Halomonas, in the wastewater almost disappeared, while the relative abundance of some minor genera including Pseudomonas and Bacillus increased dozens of times. Metagenomic analysis showed that 159 ARGs within 14 types were detectable in the samples, and the radiation at 500 mJ/cm(2) obviously increased their total relative abundance from 31.68 ppm to 190.78 ppm, which was supported by quantitative real time PCR. As the dominant persistent ARGs, multidrug resistance genes carried by Pseudomonas and bacitracin resistance gene bacA carried by Bacillus mainly contributed to the ARGs abundance increase. Bacterial community shift and MGEs replication induced by the radiation might drive the resistome alteration. The findings may shed new light on the mechanism behind the ultraviolet radiation effects on antibiotic resistance in wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

PubMed

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

2017-03-01

Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

Functional genomics indicate that schizophrenia may be an adult vascular-ischemic disorder

PubMed Central

Moises, H W; Wollschläger, D; Binder, H

2015-01-01

In search for the elusive schizophrenia pathway, candidate genes for the disorder from a discovery sample were localized within the energy-delivering and ischemia protection pathway. To test the adult vascular-ischemic (AVIH) and the competing neurodevelopmental hypothesis (NDH), functional genomic analyses of practically all available schizophrenia-associated genes from candidate gene, genome-wide association and postmortem expression studies were performed. Our results indicate a significant overrepresentation of genes involved in vascular function (P<0.001), vasoregulation (that is, perivascular (P<0.001) and shear stress (P<0.01), cerebral ischemia (P<0.001), neurodevelopment (P<0.001) and postischemic repair (P<0.001) among schizophrenia-associated genes from genetic association studies. These findings support both the NDH and the AVIH. The genes from postmortem studies showed an upregulation of vascular-ischemic genes (P=0.020) combined with downregulated synaptic (P=0.005) genes, and ND/repair (P=0.003) genes. Evidence for the AVIH and the NDH is critically discussed. We conclude that schizophrenia is probably a mild adult vascular-ischemic and postischemic repair disorder. Adult postischemic repair involves ND genes for adult neurogenesis, synaptic plasticity, glutamate and increased long-term potentiation of excitatory neurotransmission (i-LTP). Schizophrenia might be caused by the cerebral analog of microvascular angina. PMID:26261884

Functional genomics indicate that schizophrenia may be an adult vascular-ischemic disorder.

PubMed

Moises, H W; Wollschläger, D; Binder, H

2015-08-11

In search for the elusive schizophrenia pathway, candidate genes for the disorder from a discovery sample were localized within the energy-delivering and ischemia protection pathway. To test the adult vascular-ischemic (AVIH) and the competing neurodevelopmental hypothesis (NDH), functional genomic analyses of practically all available schizophrenia-associated genes from candidate gene, genome-wide association and postmortem expression studies were performed. Our results indicate a significant overrepresentation of genes involved in vascular function (P < 0.001), vasoregulation (that is, perivascular (P < 0.001) and shear stress (P < 0.01), cerebral ischemia (P < 0.001), neurodevelopment (P < 0.001) and postischemic repair (P < 0.001) among schizophrenia-associated genes from genetic association studies. These findings support both the NDH and the AVIH. The genes from postmortem studies showed an upregulation of vascular-ischemic genes (P = 0.020) combined with downregulated synaptic (P = 0.005) genes, and ND/repair (P = 0.003) genes. Evidence for the AVIH and the NDH is critically discussed. We conclude that schizophrenia is probably a mild adult vascular-ischemic and postischemic repair disorder. Adult postischemic repair involves ND genes for adult neurogenesis, synaptic plasticity, glutamate and increased long-term potentiation of excitatory neurotransmission (i-LTP). Schizophrenia might be caused by the cerebral analog of microvascular angina.

Differential expression of thymic DNA repair genes in low-dose-rate irradiated AKR/J mice

PubMed Central

Bong, Jin Jong; Kang, Yu Mi; Shin, Suk Chul; Choi, Seung Jin

2013-01-01

We previously determined that AKR/J mice housed in a low-dose-rate (LDR) (137Cs, 0.7 mGy/h, 2.1 Gy) γ-irradiation facility developed less spontaneous thymic lymphoma and survived longer than those receiving sham or high-dose-rate (HDR) (137Cs, 0.8 Gy/min, 4.5 Gy) radiation. Interestingly, histopathological analysis showed a mild lymphomagenesis in the thymus of LDR-irradiated mice. Therefore, in this study, we investigated whether LDR irradiation could trigger the expression of thymic genes involved in the DNA repair process of AKR/J mice. The enrichment analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed immune response, nucleosome organization, and the peroxisome proliferator-activated receptors signaling pathway in LDR-irradiated mice. Our microarray analysis and quantitative polymerase chain reaction data demonstrated that mRNA levels of Lig4 and RRM2 were specifically elevated in AKR/J mice at 130 days after the start of LDR irradiation. Furthermore, transcriptional levels of H2AX and ATM, proteins known to recruit DNA repair factors, were also shown to be upregulated. These data suggest that LDR irradiation could trigger specific induction of DNA repair-associated genes in an attempt to repair damaged DNA during tumor progression, which in turn contributed to the decreased incidence of lymphoma and increased survival. Overall, we identified specific DNA repair genes in LDR-irradiated AKR/J mice. PMID:23820165

Molecular response of nasal mucosa to therapeutic exposure to broad-band ultraviolet radiation

PubMed Central

Mitchell, David; Paniker, Lakshmi; Sanchez, Guillermo; Bella, Zsolt; Garaczi, Edina; Szell, Marta; Hamid, Qutayba; Kemeny, Lajos; Koreck, Andrea

2010-01-01

Abstract Ultraviolet radiation (UVR) phototherapy is a promising new treatment for inflammatory airway diseases. However, the potential carcinogenic risks associated with this treatment are not well understood. UV-specific DNA photoproducts were used as biomarkers to address this issue. Radioimmunoassay was used to quantify cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts in DNA purified from two milieus: nasal mucosa samples from subjects exposed to intranasal phototherapy and human airway (EpiAirway™) and human skin (EpiDerm™) tissue models. Immunohistochemistry was used to detect CPD formation and persistence in human nasal biopsies and human tissue models. In subjects exposed to broadband ultraviolet radiation, DNA damage frequencies were determined prior to as well as immediately after treatment and at increasing times post-treatment. We observed significant levels of DNA damage immediately after treatment and efficient removal of the damage within a few days. No residual damage was observed in human subjects exposed to multiple UVB treatments several weeks after the last treatment. To better understand the molecular response of the nasal epithelium to DNA damage, parallel experiments were conducted in EpiAirway and EpiDerm model systems. Repair rates in these two tissues were very similar and comparable to that observed in human skin. The data suggest that the UV-induced DNA damage response of respiratory epithelia is very similar to that of the human epidermis and that nasal mucosa is able to efficiently repair UVB induced DNA damage. PMID:18671762

Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted Target Locus Modifications

PubMed Central

Radecke, Sarah; Radecke, Frank; Cathomen, Toni; Schwarz, Klaus

2010-01-01

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODN-mediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation. Because I-SceI is hardly adjustable to cut at any desired position in the human genome, here, customizable zinc-finger nucleases (ZFNs) were used to stimulate ssODN-mediated repair of a mutated single-copy reporter locus stably integrated into human embryonic kidney-293 cells. The ZFNs induced faithful gene repair at a frequency of 0.16%. Six times more often, ZFN-induced DSBs were found to be modified by unfaithful addition of ssODN between the termini and about 60 times more often by nonhomologous end joining-related deletions and insertions. Additionally, ZFN off-target activity based on binding mismatch sites at the locus of interest was detected in in vitro cleavage assays and also in chromosomal DNA isolated from treated cells. Therefore, the specificity of ZFN-induced ssODN-mediated gene repair needs to be improved, especially regarding clinical applications. PMID:20068556

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

PubMed

Li, Shisheng; Smerdon, Michael J

2004-04-02

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

A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

PubMed

Scarbrough, Peter M; Weber, Rachel Palmieri; Iversen, Edwin S; Brhane, Yonathan; Amos, Christopher I; Kraft, Peter; Hung, Rayjean J; Sellers, Thomas A; Witte, John S; Pharoah, Paul; Henderson, Brian E; Gruber, Stephen B; Hunter, David J; Garber, Judy E; Joshi, Amit D; McDonnell, Kevin; Easton, Doug F; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A; Schildkraut, Joellen M

2016-01-01

DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P < 5.09 × 10(-6)), MSH5 (P < 5.09 × 10(-6)), and BRCA2 (P = 5.70 × 10(-6)). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. ©2015 American Association for Cancer Research.

Contribution of sleep to the repair of neuronal DNA double-strand breaks: evidence from flies and mice.

PubMed

Bellesi, Michele; Bushey, Daniel; Chini, Mattia; Tononi, Giulio; Cirelli, Chiara

2016-11-10

Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs.

DNA repair variants and breast cancer risk.

PubMed

Grundy, Anne; Richardson, Harriet; Schuetz, Johanna M; Burstyn, Igor; Spinelli, John J; Brooks-Wilson, Angela; Aronson, Kristan J

2016-05-01

A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P < 0.05), although no associations were observed for other DNA repair SNPs. Interactions of six SNPs in multiple DNA repair pathways with physical activity were evident prior to correction for FDR, following which there was support for only one of the interaction terms (P < 0.05). No consistent associations between variants in DNA repair genes and breast cancer risk or their modification by breast cancer risk factors were observed. © 2016 Wiley Periodicals, Inc.

Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

PubMed Central

Andreassen, Paul R.; Ren, Keqin

2016-01-01

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

DNA repair and tumorigenesis: lessons from hereditary cancer syndromes.

PubMed

Heinen, Christopher D; Schmutte, Christoph; Fishel, Richard

2002-01-01

The discovery that alterations of the DNA mismatch repair system (MMR) were linked to the common human cancer susceptibility syndrome hereditary nonpolyposis colon cancer (HNPCC) resulted in the declaration of a third class of genes involved in tumor development. In addition to oncogenes and tumor suppressors, alterations of DNA repair genes involved in maintaining genomic stability were found to be a clear cause of tum the level of the single nucleotides or chromosomes. This observation suggested that the establishment of genomic instability, termed the Mutator Phenotype, was an important aspect of tumor development.(1,2) Since the initial identification of the human MutS homolog hMSH2 nearly a decade ago,(3,4) more links have been described between human cancers and genes involved in maintaining genomic stability. Work in recent years has revealed that DNA repair proteins may also function in signaling pathways that provoke cell cycle arrest and apoptosis. This review will focus on the genetic and biochemical functions of DNA repair genes linked to hereditary cancer predisposition characterized by genomic instability (Table 1). Interestingly, the protein products of these genes have been directly or indirectly linked to the DNA damage-induce cell cycle arrest and apoptosis. We conclude that a robust connection between DNA repair proteins and damage-induced apoptosis may be as important for tumorigenesis as their role in maintaining genome stability.

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

PubMed

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

2013-12-01

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

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

PubMed Central

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

2013-01-01

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

Cockayne syndrome and xeroderma pigmentosum

PubMed Central

Rapin, I.; Lindenbaum, Y.; Dickson, D.W.; Kraemer, K.H.; Robbins, J.H.

2015-01-01

Objectives To review genetic variants of Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive disorders of DNA repair that affect the nervous system, and to illustrate them by the first case of xeroderma pigmentosum–Cockayne syndrome (XP-CS) complex to undergo neuropathologic examination. Methods Published reports of clinical, pathologic, and molecular studies of CS, XP neurologic disease, and the XP-CS complex were reviewed, and a ninth case of XP-CS is summarized. Results CS is a multisystem disorder that causes both profound growth failure of the soma and brain and progressive cachexia, retinal, cochlear, and neurologic degeneration, with a leukodystrophy and demyelinating neuropathy without an increase in cancer. XP presents as extreme photosensitivity of the skin and eyes with a 1000-fold increased frequency of cutaneous basal and squamous cell carcinomas and melanomas and a small increase in nervous system neoplasms. Some 20% of patients with XP incur progressive degeneration of previously normally developed neurons resulting in cortical, basal ganglia, cerebellar, and spinal atrophy, cochlear degeneration, and a mixed distal axonal neuropathy. Cultured cells from patients with CS or XP are hypersensitive to killing by ultraviolet (UV) radiation. Both CS and most XP cells have defective DNA nucleotide excision repair of actively transcribing genes; in addition, XP cells have defective repair of the global genome. There are two complementation groups in CS and seven in XP. Patients with the XP-CS complex fall into three XP complementation groups. Despite their XP genotype, six of nine individuals with the XP-CS complex, including the boy we followed up to his death at age 6, had the typical clinically and pathologically severe CS phenotype. Cultured skin and blood cells had extreme sensitivity to killing by UV radiation, DNA repair was severely deficient, post-UV unscheduled DNA synthesis was reduced to less than 5%, and post-UV plasmid mutation frequency was increased. Conclusions The paradoxical lack of parallelism of phenotype to genotype is unexplained in these disorders. Perhaps diverse mutations responsible for UV sensitivity and deficient DNA repair may also produce profound failure of brain and somatic growth, progressive cachexia and premature aging, and tissue-selective neurologic deterioration by their roles in regulation of transcription and repair of endogenous oxidative DNA damage. PMID:11185579

International congress on DNA damage and repair: Book of abstracts

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

Not Available

This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar, Temperate and Tropical Environments: Differential Impacts on Damage and Repair

PubMed Central

Wong, Chiew-Yen; Teoh, Ming-Li; Phang, Siew-Moi; Lim, Phaik-Eem; Beardall, John

2015-01-01

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400–700 nm), PAR plus ultraviolet-A (320–400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280–320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (Ek) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSIIeff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming. PMID:26427046

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar, Temperate and Tropical Environments: Differential Impacts on Damage and Repair.

PubMed

Wong, Chiew-Yen; Teoh, Ming-Li; Phang, Siew-Moi; Lim, Phaik-Eem; Beardall, John

2015-01-01

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400-700 nm), PAR plus ultraviolet-A (320-400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280-320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (Ek) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSIIeff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

PubMed Central

Guffanti, Federica; Fratelli, Maddalena; Ganzinelli, Monica; Bolis, Marco; Ricci, Francesca; Bizzaro, Francesca; Chilà, Rosaria; Sina, Federica Paola; Fruscio, Robert; Lupia, Michela; Cavallaro, Ugo; Cappelletti, Maria Rosa; Generali, Daniele; Giavazzi, Raffaella; Damia, Giovanna

2018-01-01

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients. PMID:29872499

TLR9 agonists oppositely modulate DNA repair genes in tumor versus immune cells and enhance chemotherapy effects.

PubMed

Sommariva, Michele; De Cecco, Loris; De Cesare, Michelandrea; Sfondrini, Lucia; Ménard, Sylvie; Melani, Cecilia; Delia, Domenico; Zaffaroni, Nadia; Pratesi, Graziella; Uva, Valentina; Tagliabue, Elda; Balsari, Andrea

2011-10-15

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients.

PubMed

Hussien, Yousry Mostafa; Gharib, Amal F; Awad, Hanan A; Karam, Rehab A; Elsawy, Wael H

2012-02-01

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene-gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.

DNA's Encounter with Ultraviolet Light: An Instinct for Self-Preservation?

PubMed

Barlev, Adam; Sen, Dipankar

2018-02-20

Photochemical modification is the major class of environmental damage suffered by DNA, the genetic material of all free-living organisms. Photolyases are enzymes that carry out direct photochemical repair (photoreactivation) of covalent pyrimidine dimers formed in DNA from exposure to ultraviolet light. The discovery of catalytic RNAs in the 1980s led to the "RNA world hypothesis", which posits that early in evolution RNA or a similar polymer served both genetic and catalytic functions. Intrigued by the RNA world hypothesis, we set out to test whether a catalytic RNA (or a surrogate, a catalytic DNA) with photolyase activity could be contemplated. In vitro selection from a random-sequence DNA pool yielded two DNA enzymes (DNAzymes): Sero1C, which requires serotonin as an obligate cofactor, and UV1C, which is cofactor-independent and optimally uses light of 300-310 nm wavelength to repair cyclobutane thymine dimers within a gapped DNA substrate. Both Sero1C and UV1C show multiple turnover kinetics, and UV1C repairs its substrate with a quantum yield of ∼0.05, on the same order as the quantum yields of certain classes of photolyase enzymes. Intensive study of UV1C has revealed that its catalytic core consists of a guanine quadruplex (G-quadruplex) positioned proximally to the bound substrate's thymine dimer. We hypothesize that electron transfer from photoexcited guanines within UV1C's G-quadruplex is responsible for substrate photoreactivation, analogous to electron transfer to pyrimidine dimers within a DNA substrate from photoexcited flavin cofactors located within natural photolyase enzymes. Though the analogy to evolution is necessarily limited, a comparison of the properties of UV1C and Sero1C, which arose out of the same in vitro selection experiment, reveals that although the two DNAzymes comparably accelerate the rate of thymine dimer repair, Sero1C has a substantially broader substrate repertoire, as it can repair many more kinds of pyrimidine dimers than UV1C. Therefore, the co-opting of an amino acid-like cofactor by a nucleic acid enzyme in this case contributes functional versatility rather than a greater rate enhancement. In recent work on UV1C, we have succeeded in shifting its action spectrum from the UVB into the blue region of the spectrum and determined that although it catalyzes both repair and de novo formation of thymine dimers, UV1C is primarily a catalyst for thymine dimer repair. Our work on photolyase DNAzymes has stimulated broader questions about whether analogous, purely nucleotide-based photoreactivation also occurs in double-helical DNA, the dominant form of DNA in living cells. Recently, a number of different groups have reported that this kind of repair is indeed operational in DNA duplexes, i.e., that there exist nucleotide sequences that actively protect, by way of photoreactivation (rather than by simply preventing their formation), pyrimidine dimers located proximal to them. Nucleotide-based photoreactivation thus appears to be a salient, if unanticipated, property of DNA and RNA. The phenomenon also offers pointers in the direction of how in primordial evolution-in an RNA world-early nucleic acids may have protected themselves from structural and functional damage wrought by ultraviolet light.

Base excision repair imbalance in colorectal cancer has prognostic value and modulates response to chemotherapy

PubMed Central

Leguisamo, Natalia M.; Gloria, Helena C.; Kalil, Antonio N.; Martins, Talita V.; Azambuja, Daniel B.

2017-01-01

Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC. PMID:28903334

Epigenetic alterations mediate iPSC normalization of DNA-repair expression and TNR stability in Huntington's disease.

PubMed

Mollica, Peter A; Zamponi, Martina; Reid, John A; Sharma, Deepak K; White, Alyson E; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

2018-06-13

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction during pluripotency and neurodevelopment, are poorly understood. Here we tested the hypothesis that hypomethylation during cellular reprogramming leads to up-regulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early-stage HD-affected neural stem cells (NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced during pluripotency, and maintained in HD-NSCs. We also identify up-regulation of 5-hmC catalyzing ten-eleven translocation (TET1/2) proteins, and show their knockdown leads to a corresponding decrease in select DNA repair gene expression. We further confirm decreased expression of TET regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms involved in pluripotency recover the selected DNA repair gene expression and stabilizes pathogenic TNRs in HD. © 2018. Published by The Company of Biologists Ltd.

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae.

PubMed

Rhee, Jae-Sung; Kim, Bo-Mi; Kim, Ryeo-Ok; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

2013-09-15

To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4Gy of radiation, and biochemical and molecular damage became substantial from 8Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae. Copyright © 2013 Elsevier B.V. All rights reserved.

Nucleotide excision repair by dual incisions in plants.

PubMed

Canturk, Fazile; Karaman, Muhammet; Selby, Christopher P; Kemp, Michael G; Kulaksiz-Erkmen, Gulnihal; Hu, Jinchuan; Li, Wentao; Lindsey-Boltz, Laura A; Sancar, Aziz

2016-04-26

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.

Cell damage caused by ultraviolet B radiation in the desert cyanobacterium Phormidium tenue and its recovery process.

PubMed

Wang, Gaohong; Deng, Songqiang; Liu, Jiafeng; Ye, Chaoran; Zhou, Xiangjun; Chen, Lanzhou

2017-10-01

Phormidium tenue, a cyanobacterium that grows in the topsoil of biological soil crusts (BSCs), has the highest recovery rate among desert crust cyanobacteria after exposure to ultraviolet B (UV-B) radiation. However, the mechanism underlying its recovery process is unclear. To address this issue, we measured chlorophyll a fluorescence, generation of reactive oxygen species (ROS), lipid peroxidation, and repair of DNA breakage in P. tenue following exposure to UV-B. We found that UV-B radiation at all doses tested reduced photosynthesis and induced cell damage in P. tenue. However, P. tenue responded to UV-B radiation by rapidly reducing photosynthetic activity, which protects the cell by leaking less ROS. Antioxidant enzymes, DNA damage repair systems, and UV absorbing pigments were then induced to mitigate the damage caused by UV-B radiation. The addition of exogenous antioxidant chemicals ascorbate and N-acetylcysteine also mitigated the harmful effects caused by UV-B radiation and enhanced the recovery process. These chemicals could aid in the resistance of P. tenue to the exposure of intense UV-B radiation in desertified areas when inoculated onto the sand surface to form artificial algal crusts. Copyright © 2017. Published by Elsevier Inc.

Requirement of the Saccharomyces cerevisiae APN1 Gene for the Repair of Mitochondrial DNA Alkylation Damage

PubMed Central

Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.

2010-01-01

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988

Response of growth and photosynthesis of Emiliania huxleyi to visible and UV irradiances under different light regimes.

PubMed

Xing, Tao; Gao, Kunshan; Beardall, John

2015-01-01

Microalgae are capable of acclimating to changes in light and ultraviolet radiation (UVR, 280-400 nm). However, little is known about how the ecologically important coccolithophore Emiliania huxleyi responds to UVR when acclimated to different light regimes. Here, we grew E. huxleyi under indoor constant light or fluctuating sunlight with or without UVR, and investigated its growth, photosynthetic performance and pigmentation. Under the indoor constant light regime, the specific growth rate (μ) was highest, while fluctuating outdoor solar radiation significantly decreased the growth rate. Addition of UVR further decreased the growth rate. The repair rate of photosystem II (PSII), as reflected in changes in PSII quantum yield, showed an inverse correlation with growth rate. Cells grown under the indoor constant light regime exhibited the lowest repair rate, while cells from the outdoor fluctuating light regimes significantly increased their repair rate. Addition of UVR increased both the repair rate and intracellular UV-absorbing compounds. This increased repair capability, at the cost of decreased growth rate, persisted after the cells were transferred back to the indoor again, suggesting an enhanced allocation of energy and resources for repair of photosynthetic machinery damage by solar UVR which persisted for a period after transfer from solar UVR. © 2014 The American Society of Photobiology.

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

DTIC Science & Technology

2012-03-01

Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2...repair pathway comprising at least 15 gene products. Mutation of any of these genes causes the human disease Fanconi anemia (FA), which is associated...genetic deficiency in components of the Fanconi anemia (FA) pathway (Wang, 2007). Cells from FA patients, or knockout mice with deficiencies in the FA

The Human L1 Element Causes DNA Double-Strand Breaks in Breast Cancer

DTIC Science & Technology

2006-08-01

cancer is complex. However, defects in DNA repair genes in the double-strand break repair pathway are cancer predisposing. My lab has characterized...a new potentially important source of double-strand breaks (DSBs) in human cells and are interested in characterizing which DNA repair genes act on...this particular source of DNA damage. Selfish DNA accounts for 45% of the human genome. We have recently demonstrated that one particular selfish

Chromosomal location and genetic mapping of the mismatch repair gene homologs MSH2, MSH3, and MSH6 in rye and wheat

PubMed

Korzun; Borner; Siebert; Malyshev; Hilpert; Kunze; Puchta

1999-12-01

The efficiency of homeologous recombination is influenced by mismatch repair genes in bacteria, yeast, and mammals. To elucidate a possible role of these genes in homeologous pairing and cross-compatibility in plants, gene probes of wheat (Triticum aestivum) specific for the mismatch repair gene homologues MSH2, MSH3, and MSH6 were used to map them to their genomic positions in rye (Secale cereale). Whereas MSH2 was mapped to the short arm of chromosome 1R, MSH3 was mapped to the long arm of chromosome 2R and MSH6 to the long arm of chromosome 5R. Southern blots with nullisomic-tetrasomic (NT) lines of wheat indicated the presence of the sequences on the respective homeologous group of wheat chromosomes. Additionally, an MSH6-specific homologue could also be detected on homoeologous group 3 of wheat. However, in the well-known, highly homoeologous pairing wheat mutant ph1b the MSH6-specific sequence is not within the deleted part of chromosome 5BL, indicating that the pairing phenotype is not due to a loss of one of the mismatch repair genes tested.

Ultraviolet-B-induced DNA damage and ultraviolet-B tolerance mechanisms in species with different functional groups coexisting in subalpine moorlands.

PubMed

Wang, Qing-Wei; Kamiyama, Chiho; Hidema, Jun; Hikosaka, Kouki

2016-08-01

High doses of ultraviolet-B (UV-B; 280-315 nm) radiation can have detrimental effects on plants, and especially damage their DNA. Plants have DNA repair and protection mechanisms to prevent UV-B damage. However, it remains unclear how DNA damage and tolerance mechanisms vary among field species. We studied DNA damage and tolerance mechanisms in 26 species with different functional groups coexisting in two moorlands at two elevations. We collected current-year leaves in July and August, and determined accumulation of cyclobutane pyrimidine dimer (CPD) as UV-B damage and photorepair activity (PRA) and concentrations of UV-absorbing compounds (UACs) and carotenoids (CARs) as UV-B tolerance mechanisms. DNA damage was greater in dicot than in monocot species, and higher in herbaceous than in woody species. Evergreen species accumulated more CPDs than deciduous species. PRA was higher in Poaceae than in species of other families. UACs were significantly higher in woody than in herbaceous species. The CPD level was not explained by the mechanisms across species, but was significantly related to PRA and UACs when we ignored species with low CPD, PRA and UACs, implying the presence of another effective tolerance mechanism. UACs were correlated negatively with PRA and positively with CARs. Our results revealed that UV-induced DNA damage significantly varies among native species, and this variation is related to functional groups. DNA repair, rather than UV-B protection, dominates in UV-B tolerance in the field. Our findings also suggest that UV-B tolerance mechanisms vary among species under evolutionary trade-off and synergism.

Hereditary Disorders with Defective Repair of UV-Induced DNA Damage

PubMed Central

Moriwaki, Shinichi

2013-01-01

Nucleotide excision repair (NER) is an essential system for correcting ultraviolet (UV)—induced DNA damage. Lesions remaining in DNA due to reduced capacity of NER may result in cellular death, premature aging, mutagenesis and carcinogenesis of the skin. So, NER is an important protection against these changes. There are three representative genodermatoses resulting from genetic defects in NER: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). In Japan, CS is similarly rare but XP is more common and TTD is less common compared to Western countries. In 1998, we established the system for the diagnosis of these disorders and we have been performing DNA repair and genetic analysis for more than 400 samples since then. At present, there is no cure for any human genetic disorder. Early diagnosis and symptomatic treatment of neurological, ocular and dermatological abnormalities should contribute to prolonging life and elevating QOL in patients. PMID:23966815

Moderate salt treatment alleviates ultraviolet-B radiation caused impairment in poplar plants

NASA Astrophysics Data System (ADS)

Ma, Xuan; Ou, Yong-Bin; Gao, Yong-Feng; Lutts, Stanley; Li, Tao-Tao; Wang, Yang; Chen, Yong-Fu; Sun, Yu-Fang; Yao, Yin-An

2016-09-01

The effects of moderate salinity on the responses of woody plants to UV-B radiation were investigated using two Populus species (Populus alba and Populus russkii). Under UV-B radiation, moderate salinity reduced the oxidation pressure in both species, as indicated by lower levels of cellular H2O2 and membrane peroxidation, and weakened the inhibition of photochemical efficiency expressed by O-J-I-P changes. UV-B-induced DNA lesions in chloroplast and nucleus were alleviated by salinity, which could be explained by the higher expression levels of DNA repair system genes under UV-B&salt condition, such as the PHR, DDB2, and MutSα genes. The salt-induced increase in organic osmolytes proline and glycine betaine, afforded more efficient protection against UV-B radiation. Therefore moderate salinity induced cross-tolerance to UV-B stress in poplar plants. It is thus suggested that woody plants growing in moderate salted condition would be less affected by enhanced UV-B radiation than plants growing in the absence of salt. Our results also showed that UV-B signal genes in poplar plants PaCOP1, PaSTO and PaSTH2 were quickly responding to UV-B radiation, but not to salt. The transcripts of PaHY5 and its downstream pathway genes (PaCHS1, PaCHS4, PaFLS1 and PaFLS2) were differently up-regulated by these treatments, but the flavonoid compounds were not involved in the cross-tolerance since their concentration increased to the same extent in both UV-B and combined stresses.

Olaparib in Treating Patients With Metastatic or Advanced Urothelial Cancer With DNA-Repair Defects

ClinicalTrials.gov

2018-06-14

Abnormal DNA Repair; ATM Gene Mutation; ATR Gene Mutation; BAP1 Gene Mutation; BARD1 Gene Mutation; BLM Gene Mutation; BRCA1 Gene Mutation; BRCA2 Gene Mutation; BRIP1 Gene Mutation; CHEK1 Gene Mutation; CHEK2 Gene Mutation; FANCC Gene Mutation; FANCD2 Gene Mutation; FANCE Gene Mutation; FANCF Gene Mutation; MEN1 Gene Mutation; Metastatic Urothelial Carcinoma; MLH1 Gene Mutation; MSH2 Gene Mutation; MSH6 Gene Mutation; MUTYH Gene Mutation; NPM1 Gene Mutation; PALB2 Gene Mutation; PMS2 Gene Mutation; POLD1 Gene Mutation; POLE Gene Mutation; PRKDC Gene Mutation; RAD50 Gene Mutation; RAD51 Gene Mutation; SMARCB1 Gene Mutation; Stage III Bladder Urothelial Carcinoma AJCC v6 and v7; Stage IV Bladder Urothelial Carcinoma AJCC v7; STK11 Gene Mutation; Urothelial Carcinoma

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens.

PubMed

Collonnier, Cécile; Guyon-Debast, Anouchka; Maclot, François; Mara, Kostlend; Charlot, Florence; Nogué, Fabien

2017-05-15

Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in. Copyright © 2017 Elsevier Inc. All rights reserved.

Lethal cellular changes induced by near ultraviolet radiation.

PubMed

Tyrrell, R M

1979-01-01

There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths. No particular DNA lesion can be linked to cell death in wild type strains. However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair. There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general. Some important specific questions remain. For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested. In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model.

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair*

PubMed Central

King, Brenee S.; Cooper, Karen L.; Liu, Ke Jian; Hudson, Laurie G.

2012-01-01

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts. PMID:23038248

Stress and DNA repair biology of the Fanconi anemia pathway

PubMed Central

Longerich, Simonne; Li, Jian; Xiong, Yong; Sung, Patrick

2014-01-01

Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care. PMID:25237197

Cloning and characterization of the mouse XPAC gene.

PubMed Central

van Oostrom, C T; de Vries, A; Verbeek, S J; van Kreijl, C F; van Steeg, H

1994-01-01

Xeroderma Pigmentosum is a human disease, which is, among others, characterized by a high incidence of (sunlight induced) skin cancer, due to a defect in nucleotide excision repair (NER). The human DNA repair gene XPAC corrects this defect in cells isolated from Xeroderma Pigmentosum complementation group A (XP-A) patients. To enable the development of a transgenic mouse model for XP-A by gene targeting in embryonic stem cells, we cloned and characterized the mouse homologue of the XPAC gene. The mouse XPAC gene was found to consist of 6 exons, spanning approximately 21 kb. The nucleotide sequence of the exons is identical to that of the also cloned the mouse XPAC cDNA. Furthermore, the deduced amino acid sequence of the XPAC protein is the same as the one published previously by Tanaka et al. From CAT assay analysis, the promoter of the XPAC gene appeared to be located within 313 bp upstream of the assumed transcriptional start site. Like the promoters of other eukaryotic DNA repair genes (i.e. ERCC-1 and XPBC/ERCC-3), the mouse XPAC promoter region lacks classical promoter elements like TATA-, GC- and CAAT boxes. However, it contains an unique polypyrimidine-rich box, which is so far only found in genes encoding DNA repair enzymes. The function of this box in the regulation of transcription is still unclear. PMID:8127648

11th International Conference of Radiation Research

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

NONE

1999-07-18

Topics discussed in the conference included the following: Radiation Physics, Radiation Chemistry and modelling--Radiation physics and dosimetry; Electron transfer in biological media; Radiation chemistry; Biophysical and biochemical modelling; Mechanisms of DNA damage; Assays of DNA damage; Energy deposition in micro volumes; Photo-effects; Special techniques and technologies; Oxidative damage. Molecular and cellular effects-- Photobiology; Cell cycle effects; DNA damage: Strand breaks; DNA damage: Bases; DNA damage Non-targeted; DNA damage: other; Chromosome aberrations: clonal; Chromosomal aberrations: non-clonal; Interactions: Heat/Radiation/Drugs; Biochemical effects; Protein expression; Gene induction; Co-operative effects; ``Bystander'' effects; Oxidative stress effects; Recovery from radiation damage. DNA damage and repair -- DNAmore » repair genes; DNA repair deficient diseases; DNA repair enzymology; Epigenetic effects on repair; and Ataxia and ATM.« less

A Preliminary Study: Human Fibroid Stro-1+/CD44+ Stem Cells Isolated From Uterine Fibroids Demonstrate Decreased DNA Repair and Genomic Integrity Compared to Adjacent Myometrial Stro-1+/CD44+ Cells.

PubMed

Prusinski Fernung, Lauren E; Al-Hendy, Ayman; Yang, Qiwei

2018-01-01

Although uterine fibroids (UFs) continue to place a major burden on female reproductive health, the mechanisms behind their origin remain undetermined. Normal myometrial stem cells may be transformed into tumor-initiating stem cells, causing UFs, due to unknown causes of somatic mutations in MED12, found in up to 85% of sporadically formed UFs. It is well established in other tumor types that defective DNA repair increases the risk of such tumorigenic somatic mutations, mechanisms not yet studied in UFs. To examine the putative cause(s) of this stem cell transformation, we analyzed DNA repair within stem cells from human UFs compared to those from adjacent myometrium to determine whether DNA repair in fibroid stem cells is compromised. Human fibroid (F) and adjacent myometrial (Myo) stem cells were isolated from fresh tissues, and gene expression relating to DNA repair was analyzed. Fibroid stem cells differentially expressed DNA repair genes related to DNA double- (DSBs) and single-strand breaks. DNA damage was measured using alkaline comet assay. Additionally, DNA DSBs were induced in these stem cells and DNA DSB repair evaluated (1) by determining changes in phosphorylation of DNA DSB-related proteins and (2) by determining differences in γ-H2AX foci formation and relative DNA repair protein RAD50 expression. Overall, F stem cells demonstrated increased DNA damage and altered DNA repair gene expression and signaling, suggesting that human F stem cells demonstrate impaired DNA repair. Compromised F stem cell DNA repair may contribute to further mutagenesis and, consequently, further growth and propagation of UF tumors.

Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.

PubMed

Ling, Kak-Ming; Sutanto, Erika N; Iosifidis, Thomas; Kicic-Starcevich, Elizabeth; Looi, Kevin; Garratt, Luke W; Martinovich, Kelly M; Lannigan, Francis J; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

2016-10-01

Evidence into the role of TGF-β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells. Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-β1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins αvβ6, αvβ8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-β1 during wound repair. TGF-β1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-β1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-β1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-β1 activators of epithelial cells, integrin αvβ6 and αvβ8 was also measured and there was no difference in αvβ6 gene expression between the two cohorts. Although integrin αvβ8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the αvβ8 mediated TGF-β1 activation was significantly downregulated. Our data has highlighted the importance of TGF-β1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells. © 2016 Asian Pacific Society of Respirology.

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

PubMed

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

2002-02-15

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

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

PubMed Central

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

2002-01-01

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (ɛ globin, p53 and γ interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to αλµοστ 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed. PMID:11842122

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

PubMed

Sia, Elaine A; Kirkpatrick, David T

2005-02-03

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

Genetic Effects of Uv Irradiation on Excision-Proficient and -Deficient Yeast during Meiosis

PubMed Central

Resnick, Michael A.; Game, John C.; Stasiewicz, Stanley

1983-01-01

The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD+) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m 2 to the rad1 strain. Exposure to 25 J/m2 had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis. PMID:6352405

Fungal Gene Mutation Analysis Elucidating Photoselective Enhancement of UV-C Disinfection Efficiency Toward Spoilage Agents on Fruit Surface.

PubMed

Zhu, Pinkuan; Li, Qianwen; Azad, Sepideh M; Qi, Yu; Wang, Yiwen; Jiang, Yina; Xu, Ling

2018-01-01

Short-wave ultraviolet (UV-C) treatment represents a potent, clean and safe substitute to chemical sanitizers for fresh fruit preservation. However, the dosage requirement for microbial disinfection may have negative effects on fruit quality. In this study, UV-C was found to be more efficient in killing spores of Botrytis cinerea in dark and red light conditions when compared to white and blue light. Loss of the blue light receptor gene Bcwcl1 , a homolog of wc-1 in Neurospora crassa , led to hypersensitivity to UV-C in all light conditions tested. The expression of Bcuve1 and Bcphr1 , which encode UV-damage endonuclease and photolyase, respectively, were strongly induced by white and blue light in a Bcwcl1 -dependent manner. Gene mutation analyses of Bcuve1 and Bcphr1 indicated that they synergistically contribute to survival after UV-C treatment. In vivo assays showed that UV-C (1.0 kJ/m 2 ) abolished decay in drop-inoculated fruit only if the UV-C treatment was followed by a dark period or red light, while in contrast, typical decay appeared on UV-C irradiated fruits exposed to white or blue light. In summary, blue light enhances UV-C resistance in B. cinerea by inducing expression of the UV damage repair-related enzymes, while the efficiency of UV-C application for fruit surface disinfection can be enhanced in dark or red light conditions; these principles seem to be well conserved among postharvest fungal pathogens.

Determination of transcriptional units and gene products from the ftsA region of Escherichia coli.

PubMed Central

Lutkenhaus, J F; Wu, H C

1980-01-01

Lambda transducing phage gamma 16-2 carries the genes envA, ftsZ, ftsA, ddl, and murC and directs the synthesis of six unique proteins in ultraviolet-irradiated cells. Various derivatives of gamma 16-2 carrying smaller segments of the bacterial deoxyribonucleic acid have also been analyzed for their capacity to direct protein synthesis in ultraviolet-irradiated cells. These results, in combination with genetic results, have allowed the gene product of each of these genes to be assigned. In addition, an unidentified gene was located counterclockwise to murC between murC and murF. Analysis of the direction of transcription indicates that murC, ddl, ftsA, and ftsZ are transcribed clockwise on the Escherichia coli genetic map, and envA is transcribed counterclockwise. In addition, it is shown that each of the genes envA, ftsZ, and ftsA can be expressed independently. Images PMID:6447690

Genetic Variation in DNA Repair Genes and Prostate Cancer Risk: Results from a Population-Based Study

PubMed Central

Agalliu, Ilir; Kwon, Erika M; Salinas, Claudia A.; Koopmeiners, Joseph S.; Ostrander, Elaine A.; Stanford, Janet L.

2009-01-01

Objective DNA repair pathways are crucial to prevent accumulation of DNA damage and maintain genomic stability. Alterations of this pathway have been reported in many cancers. An increase in oxidative DNA damage or decrease of DNA repair capacity with aging or due to germline genetic variation may affect prostate cancer risk. Methods Pooled data from two population-based studies (1,457 cases and 1,351 controls) were analyzed to examine associations between 28 SNPs in 9 DNA repair genes (APEX1, BRCA2, ERCC2, ERCC4, MGMT, MUTYH, OGG1, XPC, and XRCC1) and prostate cancer risk. We also explored whether associations varied by smoking, by family history or clinical features of prostate cancer. Results There were no associations between these SNPs and overall risk of prostate cancer. Risks did not vary either by smoking or by family history of prostate cancer. Although, two SNPs in BRCA2 (rs144848, rs1801406) and two SNPs in ERCC2 (rs1799793, rs13181) showed stronger associations with high Gleason score or advanced stage tumors when comparing homozygous men carrying the minor vs. major allele, results were not statistically significantly different between clinically aggressive and non-aggressive tumors. Conclusion Overall this study found no associations between prostate cancer and the SNPs in DNA repair genes. Given the complexity of this pathway and its crucial role in maintenance of genomic stability a pathway-based analysis of all 150 genes in DNA repair pathways, as well as exploration of gene-environment interactions may be warranted. PMID:19902366

High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

PubMed Central

St. Charles, Jordan; Hazkani-Covo, Einat; Yin, Yi; Andersen, Sabrina L.; Dietrich, Fred S.; Greenwell, Patricia W.; Malc, Ewa; Mieczkowski, Piotr; Petes, Thomas D.

2012-01-01

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote. PMID:22267500

Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

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

Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA,more » which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.« less

Melanoma prevention by MC1R selective small peptide analogs of alpha MSH | Division of Cancer Prevention

Cancer.gov

This revised application will test the hypothesis that small peptide analogs of ¿-melanocortin (¿-MSH) that are selective agonists of the melanocortin 1 receptor (MC1R) will prevent melanoma tumor formation in transgenic mouse melanoma models by enhancing repair of ultraviolet radiation (UV)-induced DNA damage and stimulating melanogenesis. We have pioneered the research on

Hypersensitivity of skin fibroblasts from basal cell nevus syndrome patients to killing by ultraviolet B but not by ultraviolet C radiation

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

Applegate, L.A.; Goldberg, L.H.; Ley, R.D.

Basal cell nevus syndrome (BCNS) is an autosomal dominant genetic disorder in which the afflicted individuals are extremely susceptible to sunlight-induced skin cancers, particularly basal cell carcinomas. However, the cellular and molecular basis for BCNS is unknown. To ascertain whether there is any relationship between genetic predisposition to skin cancer and increased sensitivity of somatic cells from BCNS patients to killing by UV radiation, we exposed skin fibroblasts established from unexposed skin biopsies of several BCNS and age- and sex-matched normal individuals to either UV-B (280-320 nm) or UV-C (254 nm) radiation and determined their survival. The results indicated thatmore » skin fibroblasts from BCNS patients were hypersensitive to killing by UV-B but not UV-C radiation as compared to skin fibroblasts from normal individuals. DNA repair studies indicated that the increased sensitivity of BCNS skin fibroblasts to killing by UV-B radiation was not due to a defect in the excision repair of pyrimidine dimers. These results indicate that there is an association between hypersensitivity of somatic cells to killing by UV-B radiation and the genetic predisposition to skin cancer in BCNS patients. In addition, these results suggest that DNA lesions (and repair processes) other than the pyrimidine dimer are also involved in the pathogenesis of sunlight-induced skin cancers in BCNS patients. More important, the UV-B sensitivity assay described here may be used as a diagnostic tool to identify presymptomatic individuals with BCNS.« less

Nuclear organization of nucleotide excision repair is mediated by RING1B dependent H2A-ubiquitylation

PubMed Central

Chitale, Shalaka; Richly, Holger

2017-01-01

One of the major cellular DNA repair pathways is nucleotide excision repair (NER). It is the primary pathway for repair of various DNA lesions caused by exposure to ultraviolet (UV) light, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts. Although lesion-containing DNA associates with the nuclear matrix after UV irradiation it is still not understood how nuclear organization affects NER. Analyzing unscheduled DNA synthesis (UDS) indicates that NER preferentially occurs in specific nuclear areas, viz the nucleolus. Upon inducing localized damage, we observe migration of damaged DNA towards the nucleolus. Employing a LacR-based tethering system we demonstrate that H2A-ubiquitylation via the UV-RING1B complex localizes chromatin close to the nucleolus. We further show that the H2A-ubiquitin binding protein ZRF1 resides in the nucleolus, and that it anchors ubiquitylated chromatin along with XPC. Our data thus provide insight into the sub-nuclear organization of NER and reveal a novel role for histone H2A-ubiquitylation. PMID:28416769

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

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

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein.more » Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.« less

Commentary on "Inherited DNA-repair gene mutations in men with metastatic prostate cancer". Pritchard CC, Mateo J, Walsh MF, De Sarkar N, Abida W, Beltran H, Garofalo A, Gulati R, Carreira S, Eeles R, Elemento O, Rubin MA, Robinson D, Lonigro R, Hussain M, Chinnaiyan A, Vinson J, Filipenko J, Garraway L, Taplin ME, AlDubayan S, Han GC, Beightol M, Morrissey C, Nghiem B, Cheng HH, Montgomery B, Walsh T, Casadei S, Berger M, Zhang L, Zehir A, Vijai J, Scher HI, Sawyers C, Schultz N, Kantoff PW, Solit D, Robson M, Van Allen EM, Offit K, de Bono J, Nelson PS. N Engl J Med. 2016;375(5):443-53.

PubMed

Freedland, Stephen J; Aronson, William J

2017-08-01

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Commentary on: "Inherited DNA-repair gene mutations in men with metastatic prostate cancer." Pritchard CC, Mateo J, Walsh MF, De Sarkar N, Abida W, Beltran H, Garofalo A, Gulati R, Carreira S, Eeles R, Elemento O, Rubin MA, Robinson D, Lonigro R, Hussain M, Chinnaiyan A, Vinson J, Filipenko J, Garraway L, Taplin ME, AlDubayan S, Han GC, Beightol M, Morrissey C, Nghiem B, Cheng HH, Montgomery B, Walsh T, Casadei S, Berger M, Zhang L, Zehir A, Vijai J, Scher HI, Sawyers C, Schultz N, Kantoff PW, Solit D, Robson M, Van Allen EM, Offit K, de Bono J, Nelson PS. N Engl J Med. 2016 Aug 4;375(5):443-53.

PubMed

Lee, Byron H

2017-09-01

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.). Copyright © 2017 Elsevier Inc. All rights reserved.

The Mechanism of Gene Targeting in Human Somatic Cells

PubMed Central

Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A.

2014-01-01

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells. PMID:24699519

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

PubMed Central

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

PubMed

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

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

PubMed

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

2014-04-21

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

The RAD24 (= Rs1) Gene Product of Saccharomyces cerevisiae Participates in Two Different Pathways of DNA Repair

PubMed Central

Eckardt-Schupp, Friederike; Siede, Wolfram; Game, John C.

1987-01-01

The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways. PMID:3549445

The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression.

PubMed

Edelmann, W; Umar, A; Yang, K; Heyer, J; Kucherlapati, M; Lia, M; Kneitz, B; Avdievich, E; Fan, K; Wong, E; Crouse, G; Kunkel, T; Lipkin, M; Kolodner, R D; Kucherlapati, R

2000-02-15

Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3-/- mice are defective in repair of insertion/ deletion mismatches but can repair base-base mismatches. Msh3-/- mice develop tumors at a late age. When the Msh3-/- and Msh6-/- mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2-/- or Mlh1-/- mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.

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

PubMed

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

2015-05-01

Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury. © 2015 by the Society for Experimental Biology and Medicine.

The germline variants in DNA repair genes in pediatric medulloblastoma: a challenge for current therapeutic strategies.

PubMed

Trubicka, Joanna; Żemojtel, Tomasz; Hecht, Jochen; Falana, Katarzyna; Piekutowska-Abramczuk, Dorota; Płoski, Rafał; Perek-Polnik, Marta; Drogosiewicz, Monika; Grajkowska, Wiesława; Ciara, Elżbieta; Moszczyńska, Elżbieta; Dembowska-Bagińska, Bożenna; Perek, Danuta; Chrzanowska, Krystyna H; Krajewska-Walasek, Małgorzata; Łastowska, Maria

2017-04-04

The defects in DNA repair genes are potentially linked to development and response to therapy in medulloblastoma. Therefore the purpose of this study was to establish the spectrum and frequency of germline variants in selected DNA repair genes and their impact on response to chemotherapy in medulloblastoma patients. The following genes were investigated in 102 paediatric patients: MSH2 and RAD50 using targeted gene panel sequencing and NBN variants (p.I171V and p.K219fs*19) by Sanger sequencing. In three patients with presence of rare life-threatening adverse events (AE) and no detected variants in the analyzed genes, whole exome sequencing was performed. Based on combination of molecular and immunohistochemical evaluations tumors were divided into molecular subgroups. Presence of variants was tested for potential association with the occurrence of rare life-threatening AE and other clinical features. We have identified altogether six new potentially pathogenic variants in MSH2 (p.A733T and p.V606I), RAD50 (p.R1093*), FANCM (p.L694*), ERCC2 (p.R695C) and EXO1 (p.V738L), in addition to two known NBN variants. Five out of twelve patients with defects in either of MSH2, RAD50 and NBN genes suffered from rare life-threatening AE, more frequently than in control group (p = 0.0005). When all detected variants were taken into account, the majority of patients (8 out of 15) suffered from life-threatening toxicity during chemotherapy. Our results, based on the largest systematic study performed in a clinical setting, provide preliminary evidence for a link between defects in DNA repair genes and treatment related toxicity in children with medulloblastoma. The data suggest that patients with DNA repair gene variants could need special vigilance during and after courses of chemotherapy.

DNA repair in Chromobacterium violaceum.

PubMed

Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

2004-03-31

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer.

PubMed

Esteban-Jurado, Clara; Franch-Expósito, Sebastià; Muñoz, Jenifer; Ocaña, Teresa; Carballal, Sabela; López-Cerón, Maria; Cuatrecasas, Miriam; Vila-Casadesús, Maria; Lozano, Juan José; Serra, Enric; Beltran, Sergi; Brea-Fernández, Alejandro; Ruiz-Ponte, Clara; Castells, Antoni; Bujanda, Luis; Garre, Pilar; Caldés, Trinidad; Cubiella, Joaquín; Balaguer, Francesc; Castellví-Bel, Sergi

2016-10-01

Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC.

Mutational Analysis of Mismatch Repair Genes, hMLH1 and hMSH2, in Sporadic Endometrial Carcinomas with Microsatellite Instability

PubMed Central

Kobayashi, Kanji; Matsushima, Mieko; Koi, Sumiko; Saito, Hiroko; Sagae, Satoru; Kudo, Ryuichi

1996-01-01

Microsatellite instability, monitored by replication error (RER), bas been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch‐repair system. We previously reported that nearly one‐quarter of sporadic endometrial carcinomas examined revealed an RER‐positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch‐repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch‐repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch‐repair system. PMID:8609062

Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability.

PubMed

Johnson, R E; Kovvali, G K; Prakash, L; Prakash, S

1996-03-29

Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer.

PubMed

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2008-05-01

The mismatch repair system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in MMR proteins have a 10(2) to 10(3)-fold increase in the mutation rate. Single nucleotide polymorphisms of mismatch repair genes have been shown to cause a decrease in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer and p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. DNA samples from 110 patients with prostate cancer and 110 healthy controls were analyzed by single strand conformational polymorphism and polymerase chain reaction-restriction fragment length polymorphism to determine the genotypic frequency of 5 polymorphic loci on 2 MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare genotype frequency between patients and controls. A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR 1.87, 95% CI 1.0-3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR 1.57, 95% CI 0.92-2.72). In p53 codon72 Arg/Pro + Pro/Pro carriers the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR 2.1, 95% CI 1.05-4.34). To our knowledge this is the first report of the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer.

Effects of early life exposure to ultraviolet C radiation on mitochondrial DNA content, transcription, ATP production, and oxygen consumption in developing Caenorhabditis elegans

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) is present in multiple copies per cell and undergoes dramatic amplification during development. The impacts of mtDNA damage incurred early in development are not well understood, especially in the case of types of mtDNA damage that are irreparable, such as ultraviolet C radiation (UVC)-induced photodimers. Methods We exposed first larval stage nematodes to UVC using a protocol that results in accumulated mtDNA damage but permits nuclear DNA (nDNA) repair. We then measured the transcriptional response, as well as oxygen consumption, ATP levels, and mtDNA copy number through adulthood. Results Although the mtDNA damage persisted to the fourth larval stage, we observed only a relatively minor ~40% decrease in mtDNA copy number. Transcriptomic analysis suggested an inhibition of aerobic metabolism and developmental processes; mRNA levels for mtDNA-encoded genes were reduced ~50% at 3 hours post-treatment, but recovered and, in some cases, were upregulated at 24 and 48 hours post-exposure. The mtDNA polymerase γ was also induced ~8-fold at 48 hours post-exposure. Moreover, ATP levels and oxygen consumption were reduced in response to UVC exposure, with marked reductions of ~50% at the later larval stages. Conclusions These results support the hypothesis that early life exposure to mitochondrial genotoxicants could result in mitochondrial dysfunction at later stages of life, thereby highlighting the potential health hazards of time-delayed effects of these genotoxicants in the environment. PMID:23374645

Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

PubMed

Keeney, Michael; Chung, Michael T; Zielins, Elizabeth R; Paik, Kevin J; McArdle, Adrian; Morrison, Shane D; Ransom, Ryan C; Barbhaiya, Namrata; Atashroo, David; Jacobson, Gunilla; Zare, Richard N; Longaker, Michael T; Wan, Derrick C; Yang, Fan

2016-08-01

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016. © 2016 Wiley Periodicals, Inc.

Polymorphisms in RAI and in genes of nucleotide and base excision repair are not associated with risk of testicular cancer.

PubMed

Laska, Magdalena J; Nexø, Bjørn A; Vistisen, Kirsten; Poulsen, Henrik Enghusen; Loft, Steffen; Vogel, Ulla

2005-07-28

Testicular cancer has been suggested to be primed in utero and there is familiar occurrence, particularly brothers and sons of men with testicular cancer have increased risk. Although no specific causative genotoxic agents have been identified, variations in DNA repair capacity could be associated with the risk of testicular cancer. A case-control study of 184 testicular cancer cases and 194 population-based controls living in the Copenhagen Greater Area in Denmark was performed. We found that neither polymorphisms in several DNA repair genes nor alleles of several polymorphisms in the chromosomal of region 19q13.2-3, encompassing the genes ASE, ERCC1, RAI and XPD, were associated with risk of testicular cancer in Danish patients. This is in contrast to other cancers, where we reported strong associations between polymorphisms in ERCC1, ASE and RAI and occurrence of basal cell carcinoma, breast cancer and lung. To our knowledge this is the first study of DNA repair gene polymorphisms and risk of testicular cancer.

Application of CRISPR/Cas9 genome editing to the study and treatment of disease.

PubMed

Pellagatti, Andrea; Dolatshad, Hamid; Valletta, Simona; Boultwood, Jacqueline

2015-07-01

CRISPR/Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The CRISPR/Cas9 method has been engineered from the type II prokaryotic CRISPR system and uses a single-guide RNA to target the Cas9 nuclease to a specific genomic sequence. Cas9 induces double-stranded DNA breaks which are repaired either by imperfect non-homologous end joining to generate insertions or deletions (indels) or, if a repair template is provided, by homology-directed repair. Due to its specificity, simplicity and versatility, the CRISPR/Cas9 system has recently emerged as a powerful tool for genome engineering in various species. This technology can be used to investigate the function of a gene of interest or to correct gene mutations in cells via genome editing, paving the way for future gene therapy approaches. Improvements to the efficiency of CRISPR repair, in particular to increase the rate of gene correction and to reduce undesired off-target effects, and the development of more effective delivery methods will be required for its broad therapeutic application.

Targeted gene therapy and cell reprogramming in Fanconi anemia

PubMed Central

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

PubMed

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

Synthetic lethality in DNA repair network: A novel avenue in targeted cancer therapy and combination therapeutics.

PubMed

Bhattacharjee, Sonali; Nandi, Saikat

2017-12-01

Synthetic lethality refers to a lethal phenotype that results from the simultaneous disruptions of two genes, while the disruption of either gene alone is viable. Many DNA double strand break repair (DSBR) genes have synthetic lethal relationships with oncogenes and tumor suppressor genes, which can be exploited for targeted cancer therapy, an approach referred to as combination therapy. DNA double-strand breaks (DSBs) are one of the most toxic lesions to a cell and can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR and NHEJ genes are particularly attractive targets for cancer therapy because these genes have altered expression patterns in cancer cells when compared with normal cells and these genetic abnormalities can be targeted for selectively killing cancer cells. Here, we review recent advances in the development of small molecule inhibitors against HR and NHEJ genes to induce synthetic lethality and address the future directions and clinical relevance of this approach. © 2017 IUBMB Life, 69(12):929-937, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Genetic polymorphisms in 85 DNA repair genes and bladder cancer risk.

PubMed

Michiels, Stefan; Laplanche, Agnès; Boulet, Thomas; Dessen, Philippe; Guillonneau, Bertrand; Méjean, Arnaud; Desgrandchamps, François; Lathrop, Mark; Sarasin, Alain; Benhamou, Simone

2009-05-01

Several defense mechanisms have been developed and maintained during the evolution to protect human cells against damage produced from exogenous or endogenous sources. We examined the associations between bladder cancer and a panel of 652 polymorphisms from 85 genes involved in maintenance of genetic stability [base excision repair, nucleotide excision repair, double-strand break repair (DSBR) and mismatch repair, as well as DNA synthesis and cell cycle regulation pathways] in 201 incident bladder cancer cases and 326 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value the expected proportion of false discoveries (q-value). Haplotype analysis revealed significant associations (P < 0.01) between bladder cancer and two genes (POLB and FANCA) with an associated q-value of 24%. A permutation test was also used to determine whether, in each pathway analyzed, there are more variants whose allelic frequencies are different between cases and controls as compared with what would be expected by chance. Differences were found for cell cycle regulation (P = 0.02) and to a lesser extent for DSBR (P = 0.05) pathways. These results hint to a few potential candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations. It is anticipated that genome-wide association studies will open new perspectives for interpretation of the results of extensive candidate gene studies such as ours.

Ultraviolet Curable Resin System for Rapid Runway Repair.

DTIC Science & Technology

1983-04-01

Diaryllodonium Ilt Decomposition upon It Expost - 2 Tiarylsulfonium salts react in the same manner as diaryliodonium salts upon UV exposure. A...diphenyl iodonium he:cafiuoroarsenate, the principal diaryliodonium salt used in this study , is approximately $2,329 per pound based solelyupon the cost of...experiments of this program. This choice was predominantly based on the fact that a study has been made on the effect of sensitizers in enhancing the UV

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals.

PubMed

Spampinato, Claudia P

2017-05-01

The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.

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

PubMed Central

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

1997-01-01

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

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2016-01-01

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings indicate that not all cyanobacteria have an E. coli-type SOS system. Also interestingly, several cyanobacteria possess multiple copies of E. coli-like DNA repair genes, such as Acaryochloris marina MBIC11017 (2 alkB, 3 ogt, 7 recA, 3 recD, 2 ssb, 3 umuC, 4 umuD, and 8 xerC), Cyanothece ATCC51142 (2 lexA and 4 ruvC), and Nostoc PCC7120 (2 ssb and 3 xerC). PMID:27881980

EGR1 induces tenogenic differentiation of tendon stem cells and promotes rabbit rotator cuff repair.

PubMed

Tao, Xu; Liu, Junpeng; Chen, Lei; Zhou, You; Tang, Kanglai

2015-01-01

The rate of healing failure after surgical repair of chronic rotator cuff tears is considerably high. The aim of this study was to investigate the function of the zinc finger transcription factor early growth response 1 (EGR1) in the differentiation of tendon stem cells (TSCs) and in tendon formation, healing, and tendon tear repair using an animal model of rotator cuff repair. Tenocyte, adipocyte, osteocyte, and chondrocyte differentiation as well as the expression of related genes were determined in EGR1-overexpressing TSCs (EGR1-TSCs) using tissue-specific staining, immunofluorescence staining, quantitative PCR, and western blotting. A rabbit rotator cuff repair model was established, and TSCs and EGR1-TSCs in a fibrin glue carrier were applied onto repair sites. The rabbits were sacrificed 8 weeks after repair operation, and tissues were histologically evaluated and tenocyte-related gene expression was determined. EGR1 induced tenogenic differentiation of TSCs and inhibited non-tenocyte differentiation of TSCs. Furthermore, EGR1 promoted tendon repair in a rabbit model of rotator cuff injury. The BMP12/Smad1/5/8 signaling pathway was involved in EGR1-induced tenogenic differentiation and rotator cuff tendon repair. EGR1 plays a key role in tendon formation, healing, and repair through BMP12/Smad1/5/8 pathway. EGR1-TSCs is a promising treatment for rotator cuff tendon repair surgeries. © 2015 S. Karger AG, Basel.

Measurement of DNA repair deficiency in workers exposed to benzene

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

Hallberg, L.M.; Au, W.W.; El Zein, R.

1996-05-01

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m{sup 2} UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repairedmore » normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m{sup 2} and 350 J/m{sup 2} were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs.« less

TGF-beta-induced early gene-1 overexpression promotes oxidative stress protection and actin cytoskeleton rearrangement in human skin fibroblasts.

PubMed

Leduc, Chloe; Sobilo, Lauren; Toumi, Hechmi; Mondon, Philippe; Lespessailles, Eric; Ossant, Fédéric; Kurfurst, Robin; Pichon, Chantal

2016-06-01

Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-β. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK). TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65±5%) and exposure to ultraviolet further increased this effect (47±3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed. TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. This study enlightens the role of TIEG-1 role in skin biology. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Resolution Mapping of Two Types of Spontaneous Mitotic Gene Conversion Events in Saccharomyces cerevisiae

PubMed Central

Yim, Eunice; O’Connell, Karen E.; St. Charles, Jordan; Petes, Thomas D.

2014-01-01

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution. PMID:24990991

Oxidized Guanine Base Lesions Function in 8-Oxoguanine DNA Glycosylase-1-mediated Epigenetic Regulation of Nuclear Factor κB-driven Gene Expression*

PubMed Central

Pan, Lang; Hao, Wenjing; Ba, Xueqing

2016-01-01

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression. PMID:27756845

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

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

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

1994-10-01

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

Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces Cerevisiae

PubMed Central

Ivanov, E. L.; Sugawara, N.; Fishman-Lobell, J.; Haber, J. E.

1996-01-01

HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching. PMID:8849880

XRCC3 Thr241Met Polymorphism is not Associated with Lung Cancer Risk in a Romanian Population.

PubMed

Catana, Andreea; Pop, Monica; Marginean, Dragos Horea; Blaga, Ioana Cristina; Porojan, Mihai Dumitru; Popp, Radu Anghel; Pop, Ioan Victor

2016-01-01

Deoxyribonucleic Acid (DNA) repair mechanisms play a critical role in protecting the cellular genome against carcinogens. X-ray cross-complementing gene 3 (XRCC3) is involved in DNA repair and therefore certain genetic polymorphisms that occur in DNA repair genes may affect the ability to repair DNA defects and may represent a risk factor in carcinogenesis. The purpose of our study was to investigate the association between XRCC3 gene substitution of Threonine with Methionine in codon 241 of XRCC3 gene (Thr241Met) polymorphism and the risk of lung cancer, in a Romanian population. We recruited 93 healthy controls and 85 patients with lung cancer, all smokers. Thr241Met, XRCC3 gene genotyping was determined by multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis (OR, recessive model), did not revealed an increased risk for lung cancer, for the variant 241Met allele and Thr241Met genotypes (p=0.138, OR=0.634, CI=0.348-1.157; p=0.023, OR=0.257, CI=0.085-6.824). Also, there were no positive statistical associations between Thr241Met polymorphism of XRCC3 gene, gender, tobacco and various histopathological tumor type of lung cancer. In conclusion, the results of the study suggest that the XRCC3 gene Thr241Met polymorphism is not associated with an increased risk for the development of lung cancer in this Romanian group.

Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

PubMed

Arthur-Farraj, Peter J; Morgan, Claire C; Adamowicz, Martyna; Gomez-Sanchez, Jose A; Fazal, Shaline V; Beucher, Anthony; Razzaghi, Bonnie; Mirsky, Rhona; Jessen, Kristjan R; Aitman, Timothy J

2017-09-12

Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cartilage constructs engineered from chondrocytes overexpressing IGF-I improve the repair of osteochondral defects in a rabbit model.

PubMed

Madry, H; Kaul, G; Zurakowski, D; Vunjak-Novakovic, G; Cucchiarini, M

2013-04-16

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes overexpressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-overexpressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects.

CARTILAGE CONSTRUCTS ENGINEERED FROM CHONDROCYTES OVEREXPRESSING IGF-I IMPROVE THE REPAIR OF OSTEOCHONDRAL DEFECTS IN A RABBIT MODEL

PubMed Central

Madry, Henning; Kaul, Gunter; Zurakowski, David; Vunjak-Novakovic, Gordana; Cucchiarini, Magali

2015-01-01

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes over expressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-over expressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects. PMID:23588785

COMPREHENSIVE ANALYSES OF DNA REPAIR PATHWAYS, SMOKING, AND BLADDER CANCER RISK IN LOS ANGELES AND SHANGHAI

PubMed Central

Corral, Roman; Lewinger, Juan Pablo; Berg, David Van Den; Joshi, Amit D.; Yuan, Jian-Min; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Pike, Malcolm C.; Conti, David V.; Thomas, Duncan C.; Edlund, Christopher K.; Gao, Yu-Tang; Xiang, Yong-Bing; Zhang, Wei; Su, Yu-Chen; Stern, Mariana C.

2014-01-01

Tobacco smoking is a bladder cancer risk factor and a source of carcinogens that induce DNA damage to urothelial cells. Using data and samples from 988 cases and 1,004 controls enrolled in the Los Angeles County Bladder Cancer Study and the Shanghai Bladder Cancer Study we investigated associations between bladder cancer risk and 632 tagSNPs that comprehensively capture genetic variation in 28 DNA repair genes from four DNA repair pathways: base excision repai, nucleotide excision repair (NER), non-homologous end-joining (NHEJ), and homologous recombination repair (HHR). Odds ratios (ORs) and 95% confidence intervals (CIs) for each tagSNP were corrected for multiple testing for all SNPs within each gene using pACT, and for genes within each pathway and across pathways with Bonferroni. Gene and pathway summary estimates were obtained using ARTP. We observed an association between bladder cancer and POLB rs7832529 (BER) (pACT = 0.003; ppathway = 0.021) among all, and SNPs in XPC (NER) and OGG1 (BER) among Chinese men and women, respectively. The NER pathway showed an overall association with risk among Chinese males (ARTP NER p = 0.034). The XRCC6 SNP rs2284082 (NHEJ), also in LD with SREBF2, showed an interaction with smoking (Smoking status interaction pgene = 0.001, ppathway = 0.008, poverall = 0.034). Our findings support a role in bladder carcinogenesis for regions that map close to or within BER (POLB, OGG1) and NER genes (XPC). A SNP that tags both the XRCC6 and SREBF2 genes strongly modifies the association between bladder cancer risk and smoking. PMID:24382701

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

PubMed Central

Hadjiargyrou, Michael; O’Keefe, Regis J

2015-01-01

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

Space Shuttle Projects

NASA Image and Video Library

1984-04-01

This is a photograph of the free-flying Solar Maximum Mission Satellite (SMMS), or Solar Max, as seen by the approaching Space Shuttle Orbiter Challenger STS-41C mission. Launched April 6, 1984, one of the goals of the STS-41C mission was to repair the damaged Solar Max. The original plan was to make an excursion out to the SMMS for capture to make necessary repairs, however, this attempted feat was unsuccessful. It was necessary to capture the satellite via the orbiter's Remote Manipulator System (RMS) and secure it into the cargo bay in order to perform the repairs, which included replacing the altitude control system and the coronograph/polarimeter electronics box. The SMMS was originally launched into space via the Delta Rocket in February 1980, with the purpose to provide a means of studying solar flares during the most active part of the current sunspot cycle. Dr. Einar Tandberg-Hanssen of Marshall Space Flight Center's Space Sciences Lab was principal investigator for the Ultraviolet Spectrometer and Polarimeter, one of the seven experiments on the Solar Max.

Space Shuttle Projects

NASA Image and Video Library

1984-04-01

Launched April 6, 1984, one of the goals of the STS-41C mission was to repair the damaged free-flying Solar Maximum Mission Satellite (SMMS), or Solar Max. The original plan was to make an excursion out to the SMMS and capture it for necessary repairs. Pictured is Mission Specialist George Nelson approaching the damaged satellite in a capture attempt. This attempted feat was unsuccessful. It was necessary to capture the satellite via the orbiter's Remote Manipulator System (RMS) and secure it into the cargo bay in order to perform the repairs, which included replacing the altitude control system and the coronograph/polarimeter electronics box. The SMMS was originally launched into space via the Delta Rocket in February 1980, with the purpose to provide a means of studying solar flares during the most active part of the current sunspot cycle. Dr. Einar Tandberg-Hanssen of Marshall Space Flight Center's Space Sciences Lab was principal investigator for the Ultraviolet Spectrometer and Polarimeter, one of the seven experiments of the Solar Max.

Whole-genome landscape of pancreatic neuroendocrine tumours.

PubMed

Scarpa, Aldo; Chang, David K; Nones, Katia; Corbo, Vincenzo; Patch, Ann-Marie; Bailey, Peter; Lawlor, Rita T; Johns, Amber L; Miller, David K; Mafficini, Andrea; Rusev, Borislav; Scardoni, Maria; Antonello, Davide; Barbi, Stefano; Sikora, Katarzyna O; Cingarlini, Sara; Vicentini, Caterina; McKay, Skye; Quinn, Michael C J; Bruxner, Timothy J C; Christ, Angelika N; Harliwong, Ivon; Idrisoglu, Senel; McLean, Suzanne; Nourse, Craig; Nourbakhsh, Ehsan; Wilson, Peter J; Anderson, Matthew J; Fink, J Lynn; Newell, Felicity; Waddell, Nick; Holmes, Oliver; Kazakoff, Stephen H; Leonard, Conrad; Wood, Scott; Xu, Qinying; Nagaraj, Shivashankar Hiriyur; Amato, Eliana; Dalai, Irene; Bersani, Samantha; Cataldo, Ivana; Dei Tos, Angelo P; Capelli, Paola; Davì, Maria Vittoria; Landoni, Luca; Malpaga, Anna; Miotto, Marco; Whitehall, Vicki L J; Leggett, Barbara A; Harris, Janelle L; Harris, Jonathan; Jones, Marc D; Humphris, Jeremy; Chantrill, Lorraine A; Chin, Venessa; Nagrial, Adnan M; Pajic, Marina; Scarlett, Christopher J; Pinho, Andreia; Rooman, Ilse; Toon, Christopher; Wu, Jianmin; Pinese, Mark; Cowley, Mark; Barbour, Andrew; Mawson, Amanda; Humphrey, Emily S; Colvin, Emily K; Chou, Angela; Lovell, Jessica A; Jamieson, Nigel B; Duthie, Fraser; Gingras, Marie-Claude; Fisher, William E; Dagg, Rebecca A; Lau, Loretta M S; Lee, Michael; Pickett, Hilda A; Reddel, Roger R; Samra, Jaswinder S; Kench, James G; Merrett, Neil D; Epari, Krishna; Nguyen, Nam Q; Zeps, Nikolajs; Falconi, Massimo; Simbolo, Michele; Butturini, Giovanni; Van Buren, George; Partelli, Stefano; Fassan, Matteo; Khanna, Kum Kum; Gill, Anthony J; Wheeler, David A; Gibbs, Richard A; Musgrove, Elizabeth A; Bassi, Claudio; Tortora, Giampaolo; Pederzoli, Paolo; Pearson, John V; Waddell, Nicola; Biankin, Andrew V; Grimmond, Sean M

2017-03-02

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

PT-12, a putative ras-activated proliferation-dependent gene, is expressed in patellar tendon and not in anterior cruciate ligament.

PubMed

Goomer, R S; Maris, T; Ostrander, R; Amiel, D

1999-09-01

We describe a gene (PT-12) that is expressed in the patellar tendon and not in the anterior cruciate ligament. We used a recently developed polymerase chain reaction-based subtractive cDNA analysis to discover genes that are overexpressed in the patellar tendon but not expressed in the anterior cruciate ligament; the long-term objective was to find genes that are central to the self-repair of the patellar tendon, in contrast with the inability of the anterior cruciate ligament to launch a repair response following injury. PT-12 is a homologue of human S2 or mouse LLRep3 ribosomal genes, which are known to be overexpressed in highly proliferating cells. This study opens a new vista to the development of techniques and reagents to study the differences between two periarticular tissues (i.e., the patellar tendon and anterior cruciate ligament) that differ primarily in their ability to self-repair.

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

PubMed

Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

2015-08-01

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

[Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts].

PubMed

Zakharov, I A; Kasinova, G V; Koval'tsova, S V

1983-01-01

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.

A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

PubMed Central

Nouvel, Laurent X; Vultos, Tiago Dos; Kassa-Kelembho, Eric; Rauzier, Jean; Gicquel, Brigitte

2007-01-01

Background Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). Results In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes. PMID:17506895

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

PubMed Central

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

2013-01-01

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

Ultraviolet Light Surface Treatment as an Environmentally Benign Process for Production, Maintenance and Repair of Military Composite Structures

NASA Astrophysics Data System (ADS)

Drzal, Lawrence T.

2002-02-01

The principal objective of this work is to develop a low-cost, high-speed, environmentally benign, dry surface treatment method for production, and repair of military composite structures using ultraviolet (UV) light in ambient air. The potential advantage of this method is that it would eliminate volatile organic wastes (VOCs), reduce or eliminate the use of solutions and detergents, and provide a robust surface that would enhance or eliminate the use of solutions and detergents, and provide a robust surface that would enhance the wetting and spreading of paints, coatings and adhesives on polymeric and inorganic surfaces treated by this method. A manufacturing base for UV production equipment is in place although not for this application. There is a need for development of an environmentally friendly, cost effective as well as a robust surface treatment method that can clean a surface as well as create a beneficial chemistry for painting and produce optimum adhesive bonding of polymers, polymer composites and metal surfaces. With this in mind, three main technical objectives were sought in the work. The first objective was to determine the usefulness of UV and UV/O(3) to surface treatments to clean and chemically modify the surface of typical PMCs used in DOD systems. The second objective was to determine the effectiveness of this surface preparation for production and/or repair of adhesively bonded, painted and/or coated polymer matrix composite structures. Finally, a determination of the environmental and performance benefits of this method as a new environmentally benign processing method for the production and/or re air of adhesively SERDP, SERDP collection, robust surface, polymeric surface, inorganic surface, volatile organic compounds (VOC) emissions.

Ultraviolet-induced movement of the human DNA repair protein, xeroderma pigmentosum type G, in the nucleus

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

Park, M.S.; Knauf, J.A.; Pendergrass, S.H.

1996-08-06

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. USing confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number ofmore » XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using {beta}-galactosidase-XPG fusion constructs ({beta}-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized {beta}-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic {beta}-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus. 50 refs., 5 figs., 1 tab.« less

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing.

PubMed

Certo, Michael T; Morgan, Richard A

2016-03-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing

PubMed Central

Certo, Michael T; Morgan, Richard A

2016-01-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications. PMID:26796671

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

PubMed

Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

2008-03-01

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

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

PubMed

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

2016-08-01

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

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break

PubMed Central

Harmsen, Tim; Klaasen, Sjoerd; van de Vrugt, Henri; te Riele, Hein

2018-01-01

Abstract Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3′ half of the ssODN. Furthermore, protecting the ssODN 3′ end with phosphorothioate linkages enhances MMR-dependent gene editing events. Our findings can be exploited to optimize efficiencies of nucleotide substitutions distal from the DSB and imply that oligonucleotide-mediated gene editing is effectuated by templated break repair. PMID:29447381

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair.

PubMed

Sterpone, Silvia; Cozzi, Renata

2010-07-25

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

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

PubMed Central

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

2014-01-01

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

The Intertwined Roles of Transcription and Repair Proteins

PubMed Central

Fong, Yick W.; Cattoglio, Claudia; Tjian, Robert

2014-01-01

Transcription is apparently risky business. Its intrinsic mutagenic potential must be kept in check by networks of DNA repair factors that monitor the transcription process to repair DNA lesions that could otherwise compromise transcriptional fidelity and genome integrity. Intriguingly, recent studies point to an even more direct function of DNA repair complexes as co-activators of transcription and the unexpected role of “scheduled” DNA damage/repair at gene promoters. Paradoxically, spontaneous DNA double-strand breaks also induce ectopic transcription that is essential for repair. Thus, transcription, DNA damage and repair may be more physically and functionally intertwined than previously appreciated. PMID:24207023

The Cell's Sophisticated Army to Defend Against Assaults on DNAThe Cell's Sophisticated Army to Defend Against Assaults on DNA | Center for Cancer Research

Cancer.gov

The maintenance of genome integrity and function is essen-tial for the survival of cells and organisms. Any damage to our genetic material must be immediately sensed and repaired to preserve a cell’s func-tional integrity. Cells are constantly faced with the challenge of protecting their DNA from assaults by damaging chemicals and ultraviolet light. DNA damage that escapes

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

PubMed

Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

2012-09-01

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

True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

PubMed Central

Le May, Nicolas; Egly, Jean-Marc; Coin, Frédéric

2010-01-01

Nucleotide excision repair (NER) is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α) and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process. PMID:20725631

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

NASA Astrophysics Data System (ADS)

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

2014-11-01

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

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

Transcriptional profiling reveals elevated Sox2 in DNA polymerase ß null mouse embryonic fibroblasts

PubMed Central

Li, Jianfeng; Luthra, Soumya; Wang, Xiao-Hong; Chandran, Uma R; Sobol, Robert W

2012-01-01

There are over 150 human proteins that have been categorized as bona fide DNA repair proteins. These DNA repair proteins maintain the integrity of the genome, reducing the onset of cancer, disease and aging phenotypes. Variations in expression and/or function would therefore impact genome integrity as well as the cellular response to genotoxins. Global gene expression analysis is an effective approach to uncover defects in DNA repair gene expression and to discover cellular and/or organismal effects brought about by external stimuli such as environmental genotoxicants, chemotherapeutic regimens, viral infections as well as developmental and age-related stimuli. Given the significance of genome stability in cell survival and response to stimuli, we have hypothesized that cells may undergo transcriptional re-programming to accommodate defects in basal DNA repair capacity to promote survival. As a test of this hypothesis, we have compared the transcriptome in three DNA polymerase ß knockout (Polß-KO) mouse embryonic fibroblasts (MEFs) and the corresponding wild-type (WT) littermate control cell lines. Each Polß-KO cell line was found to have a range of genes up-regulated, when compared to its WT littermate control cell line. Interestingly, six (6) genes were commonly up regulated in all three Polß-KO cell lines, including Sox2, one of several genes associated with the induction of pluripotent stem cells. Herein, we present these findings and suggest that loss of DNA repair and the induction of cellular transcriptional re-programming may, in part, contribute to tumor formation and the cellular response to external stimuli. PMID:23226616

Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams

PubMed Central

Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N.; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun

2013-01-01

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar ‘Sasanishiki’) that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined. PMID:23381954

Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams.

PubMed

Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun

2013-07-01

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.

Zebrafish: swimming towards a role for fanconi genes in DNA repair.

PubMed

Scata, Kimberly A; El-Deiry, Wafik S

2004-06-01

The zebrafish, Danio rerio, has become a favorite model organism for geneticists and developmental biologists. Recently cancer biologists have turned to this tiny fish to help them unravel the mysteries of conserved pathways such as the Fanconi Anemia (FA) pathway. Although a relatively rare disease, the genes involved in FA are part of a large network of DNA damage response/repair genes. Liu and colleagues have recapitulated some of the clinical manifestations of human FA by knocking down the zebrafish FANC-D2 gene thereby providing a new model for probing the underlying causes of these phenotypes.

Targeted gene therapy and cell reprogramming in Fanconi anemia.

PubMed

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-06-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Non-functional genes repaired at the RNA level.

PubMed

Burger, Gertraud

2016-01-01

Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

A novel function of adenomatous polyposis coli (APC) in regulating DNA repair

PubMed Central

Jaiswal, Aruna S.; Narayan, Satya

2008-01-01

Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase β (Pol-β) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis and chemotherapy by determining whether cells with DNA damage survive or undergo apoptosis. In this review, we summarize the evidence supporting this novel concept and suggest that these results will have implications for the development of more effective strategies for chemoprevention, prognosis, and chemotherapy of certain types of tumors. PMID:18662849

Differential DNA lesion formation and repair in heterochromatin and euchromatin

PubMed Central

Han, Chunhua; Srivastava, Amit Kumar; Cui, Tiantian; Wang, Qi-En; Wani, Altaf A.

2016-01-01

Discretely orchestrated chromatin condensation is important for chromosome protection from DNA damage. However, it is still unclear how different chromatin states affect the formation and repair of nucleotide excision repair (NER) substrates, e.g. ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts (6-4PP), as well as cisplatin-induced intrastrand crosslinks (Pt-GG). Here, by using immunofluorescence and chromatin immunoprecipitation assays, we have demonstrated that CPD, which cause minor distortion of DNA double helix, can be detected in both euchromatic and heterochromatic regions, while 6-4PP and Pt-GG, which cause major distortion of DNA helix, can exclusively be detected in euchromatin, indicating that the condensed chromatin environment specifically interferes with the formation of these DNA lesions. Mechanistic investigation revealed that the class III histone deacetylase SIRT1 is responsible for restricting the formation of 6-4PP and Pt-GG in cells, probably by facilitating the maintenance of highly condensed heterochromatin. In addition, we also showed that the repair of CPD in heterochromatin is slower than that in euchromatin, and DNA damage binding protein 2 (DDB2) can promote the removal of CPD from heterochromatic region. In summary, our data provide evidence for differential formation and repair of DNA lesions that are substrates of NER. Both the sensitivity of DNA to damage and the kinetics of repair can be affected by the underlying level of chromatin compaction. PMID:26717995

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

PubMed

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Dimer excision in Escherichia coli in the presence of caffeine

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

Rothman, R.H.

1980-07-01

The observation that polA1 and recL152 mutations result in both slow pyrimidine dimer excision and large repair patch size leads to the hypothesis that patch size is directly related to the rate of excision. In this study caffeine, a known inhibitor of excision repair, was used to examine the extent of correlation between excision rate and patch size by measuring patch size in the presence of several concentrations of caffeine. Both the rate of excision and the resistance to ultraviolet radiation were reduced with increasing concentrations of caffeine after irradiation. Caffeine also inhibited the rate at which incisions were mademore » and prolonged the time required to rejoin the discontinuities. Patch size, however, was unaffected by caffeine treatment.« less

Identification and Characterization of uvrA, a DNA Repair Gene of Deinococcus radiodurans

DTIC Science & Technology

1996-01-01

and Classificalion I 2 . TheCellWall 4 3. Intracellular Molecules 7 4. Genetics _ _ _ _ _.. 8 a. DNA COntent. 8 b. Chromosomes 8 c. Plasmids 10 d...Summary 11 B. DNA Damaging Agenls 12 I. Visible Light and Low-Frequency UV Radiation 12 2 . High-frequency UV Radiation 13 a. Pyrimidine DiIners 13 b. The...23 a. Photoreactivation Repair 23 b. Repair of Spore Pholoproducts 27 2 . Repair by Methods Involving Single Proteins 27 a. Repair of

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure and expression of the human XPBC/ERCC-3 gene involved in DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome.

PubMed Central

Weeda, G; Ma, L B; van Ham, R C; van der Eb, A J; Hoeijmakers, J H

1991-01-01

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene. Images PMID:1956789

Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases.

PubMed

Kolli, Nivya; Lu, Ming; Maiti, Panchanan; Rossignol, Julien; Dunbar, Gary L

2018-01-01

Increased accumulation of transcribed protein from the damaged DNA and reduced DNA repair capability contributes to numerous neurological diseases for which effective treatments are lacking. Gene editing techniques provide new hope for replacing defective genes and DNA associated with neurological diseases. With advancements in using such editing tools as zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs), etc., scientists are able to design DNA-binding proteins, which can make precise double-strand breaks (DSBs) at the target DNA. Recent developments with the CRISPR-Cas9 gene-editing technology has proven to be more precise and efficient when compared to most other gene-editing techniques. Two methods, non-homologous end joining (NHEJ) and homology-direct repair (HDR), are used in CRISPR-Cas9 system to efficiently excise the defective genes and incorporate exogenous DNA at the target site. In this review article, we provide an overview of the CRISPR-Cas9 methodology, including its molecular mechanism, with a focus on how in this gene-editing tool can be used to counteract certain genetic defects associated with neurological diseases. Detailed understanding of this new tool could help researchers design specific gene editing strategies to repair genetic disorders in selective neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dynamics of wound healing signaling as a potential therapeutic target for radiation-induced tissue damage.

PubMed

Chung, Yih-Lin; Pui, Newman N M

2015-01-01

We hypothesized the histone deacetylase inhibitor phenylbutyrate (PB) has beneficial effects on radiation-induced injury by modulating the expression of DNA repair and wound healing genes. Hamsters received a radiosurgical dose of radiation (40 Gy) to the cheek and were treated with varying PB dosing regimens. Gross alteration of the irradiated cheeks, eating function, histological changes, and gene expression during the course of wound healing were compared between treatment groups. Pathological analysis showed decreased radiation-induced mucositis, facilitated epithelial cell growth, and preventing ulcerative wound formation, after short-term PB treatment, but not after vehicle or sustained PB. The radiation-induced wound healing gene expression profile exhibited a sequential transition from the inflammatory and DNA repair phases to the tissue remodeling phase in the vehicle group. Sustained PB treatment resulted in a prolonged wound healing gene expression profile and delayed the wound healing process. Short-term PB shortened the duration of inflammatory cytokine expression, triggered repeated pulsed expression of cell cycle and DNA repair-regulating genes, and promoted earlier oscillatory expression of tissue remodeling genes. Distinct gene expression patterns between sustained and short-term treatment suggest dynamic profiling of wound healing gene expression can be an important part of a biological therapeutic strategy to mitigate radiation-related tissue injury. © 2015 by the Wound Healing Society.

Molecular changes preceding endometrial and ovarian cancer: a study of consecutive endometrial specimens from Lynch syndrome surveillance.

PubMed

Niskakoski, Anni; Pasanen, Annukka; Lassus, Heini; Renkonen-Sinisalo, Laura; Kaur, Sippy; Mecklin, Jukka-Pekka; Bützow, Ralf; Peltomäki, Päivi

2018-03-27

Molecular alterations preceding endometrial and ovarian cancer and the sequence of events are unknown. Consecutive specimens from lifelong surveillance for Lynch syndrome provides a natural setting to address such questions. To molecularly define the multistep gynecological tumorigenesis, DNA mismatch repair gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nation-wide registry and endometrial biopsy specimens taken from these individuals during 20 years of screening were collected. A total of 213 endometrial and ovarian specimens from Lynch syndrome individuals and 197 histology-matched (non-serous) samples from sporadic cases were available for this investigation. The specimens were profiled for markers linked to endometrial and ovarian tumorigenesis, including ARID1A protein expression, mismatch repair status, and tumor suppressor gene promoter methylation. In Lynch syndrome-associated endometrial and ovarian carcinomas, ARID1A protein was lost in 61-100% and mismatch repair was deficient in 97-100%, compared to 0-17% and 14-44% in sporadic cases (P = 0.000). ARID1A loss appeared in complex hyperplasia and deficient mismatch repair and tumor suppressor gene promoter methylation in histologically normal endometrium. Despite quantitative differences between Lynch syndrome and sporadic cases, ARID1A expression, mismatch repair, and tumor suppressor gene promoter methylation divided endometrial samples from both patient groups into three categories of increasing abnormality, comprising normal endometrium and simple hyperplasia (I), complex hyperplasia with or without atypia (II), and endometrial cancer (III). Complex hyperplasias without vs. with atypia were molecularly indistinguishable. In conclusion, surveillance specimens from Lynch syndrome identify mismatch repair deficiency, tumor suppressor gene promoter methylation, and ARID1A loss as early changes in tumor development. Our findings are clinically relevant for the classification of endometrial hyperplasias and have potential implications in cancer prevention in Lynch syndrome and beyond.

Transcriptomic responses to wounding: meta-analysis of gene expression microarray data.

PubMed

Sass, Piotr Andrzej; Dąbrowski, Michał; Charzyńska, Agata; Sachadyn, Paweł

2017-11-07

A vast amount of microarray data on transcriptomic response to injury has been collected so far. We designed the analysis in order to identify the genes displaying significant changes in expression after wounding in different organisms and tissues. This meta-analysis is the first study to compare gene expression profiles in response to wounding in as different tissues as heart, liver, skin, bones, and spinal cord, and species, including rat, mouse and human. We collected available microarray transcriptomic profiles obtained from different tissue injury experiments and selected the genes showing a minimum twofold change in expression in response to wounding in prevailing number of experiments for each of five wound healing stages we distinguished: haemostasis & early inflammation, inflammation, early repair, late repair and remodelling. During the initial phases after wounding, haemostasis & early inflammation and inflammation, the transcriptomic responses showed little consistency between different tissues and experiments. For the later phases, wound repair and remodelling, we identified a number of genes displaying similar transcriptional responses in all examined tissues. As revealed by ontological analyses, activation of certain pathways was rather specific for selected phases of wound healing, such as e.g. responses to vitamin D pronounced during inflammation. Conversely, we observed induction of genes encoding inflammatory agents and extracellular matrix proteins in all wound healing phases. Further, we selected several genes differentially upregulated throughout different stages of wound response, including established factors of wound healing in addition to those previously unreported  in this context such as PTPRC and AQP4. We found that transcriptomic responses to wounding showed similar traits in a diverse selection of tissues including skin, muscles, internal organs and nervous system. Notably, we distinguished transcriptional induction of inflammatory genes not only in the initial response to wounding, but also later, during wound repair and tissue remodelling.

Non-homologous end joining mediated DNA repair is impaired in the NUP98-HOXD13 mouse model for myelodysplastic syndrome.

PubMed

Puthiyaveetil, Abdul Gafoor; Reilly, Christopher M; Pardee, Timothy S; Caudell, David L

2013-01-01

Chromosomal translocations typically impair cell differentiation and often require secondary mutations for malignant transformation. However, the role of a primary translocation in the development of collaborating mutations is debatable. To delineate the role of leukemic translocation NUP98-HOXD13 (NHD13) in secondary mutagenesis, DNA break and repair mechanisms in stimulated mouse B lymphocytes expressing NHD13 were analyzed. Our results showed significantly reduced expression of non-homologous end joining (NHEJ)-mediated DNA repair genes, DNA Pkcs, DNA ligase4, and Xrcc4 leading to cell cycle arrest at G2/M phase. Our results showed that expression of NHD13 fusion gene resulted in impaired NHEJ-mediated DNA break repair. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biallelic PMS2 Mutation and Heterozygous DICER1 Mutation Presenting as Constitutional Mismatch Repair Deficiency With Corpus Callosum Agenesis: Case Report and Review of Literature.

PubMed

Cheyuo, Cletus; Radwan, Walid; Ahn, Janice; Gyure, Kymberly; Qaiser, Rabia; Tomboc, Patrick

2017-10-01

Constitutional mismatch repair deficiency syndrome is a cancer predisposition syndrome caused by autosomal recessive biallelic (homozygous) germline mutations in the mismatch repair genes (MLH1, MSH2, MSH6, and PMS2). The clinical spectrum includes neoplastic and non-neoplastic manifestations. We present the case of a 7-year-old boy who presented with T-lymphoblastic lymphoma and glioblastoma, together with non-neoplastic manifestations including corpus callosum agenesis, arachnoid cyst, developmental venous anomaly, and hydrocephalus. Gene mutation analysis revealed pathogenic biallelic mutations of PMS2 and heterozygous DICER1 variant predicted to be pathogenic. This report is the first to allude to a possible interaction of the mismatch repair system with DICER1 to cause corpus callosum agenesis.

Cellular responses to environmental DNA damage

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

Not Available

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.

Clinical Management and Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood.

PubMed

Tabori, Uri; Hansford, Jordan R; Achatz, Maria Isabel; Kratz, Christian P; Plon, Sharon E; Frebourg, Thierry; Brugières, Laurence

2017-06-01

Replication proofreading is crucial to avoid mutation accumulation in dividing cells. In humans, proofreading and replication repair is maintained by the exonuclease domains of DNA polymerases and the mismatch repair system. Individuals harboring germline mutations in genes involved in this process are at increased risk of early cancers from multiple organs. Biallelic mutations in any of the four mismatch repair genes MSH2, MSH6, MLH1 , and PMS2 result in one of the most aggressive childhood cancer predisposition syndromes, termed constitutional mismatch repair deficiency or constitutional mismatch repair deficiency syndrome (CMMRD). Data gathered in the last decade allow us to better define the clinical manifestations, tumor spectrum, and diagnostic algorithms for CMMRD. In this article, we summarize this information and present a comprehensive consensus surveillance protocol for these individuals. Ongoing research will allow for further definition of replication repair-deficient cancer syndromes, assessing the cost-effectiveness of such surveillance protocols and potential therapeutic interventions for these children and families. Clin Cancer Res; 23(11); e32-e37. ©2017 AACR See all articles in the online-only CCR Pediatric Oncology Series. ©2017 American Association for Cancer Research.

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases

PubMed Central

Bettencourt, Conceição; Hensman‐Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas‐Gómez, Petra; García‐Velázquez, Lizbeth Esmeralda; Alonso‐Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J.

2016-01-01

Objective The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome‐wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. Methods We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single‐nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. Results In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10–5). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10–5) and all SCAs (p = 2.22 × 10–4) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10–5), all in the same direction as in the HD GWAS. Interpretation We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983–990 PMID:27044000

Developmental and Wound-, Cold-, Desiccation-, Ultraviolet-B-Stress-Induced Modulations in the Expression of the Petunia Zinc Finger Transcription Factor Gene ZPT2-21

PubMed Central

van der Krol, Alexander R.; van Poecke, Remco M.P.; Vorst, Oscar F.J.; Voogt, Charlotte; van Leeuwen, Wessel; Borst-Vrensen, Tanja W.M.; Takatsuji, Hiroshi; van der Plas, Linus H.W.

1999-01-01

The ZPT2-2 gene belongs to the EPF gene family in petunia (Petunia hybrida), which encodes proteins with TFIIIA-type zinc-finger DNA-binding motifs. To elucidate a possible function for ZPT2-2, we analyzed its pattern of expression in relation to different developmental and physiological stress signals. The activity of the ZPT2-2 promoter was analyzed using a firefly luciferase (LUC) reporter gene, allowing for continuous measurements of transgene activity in planta. We show that ZPT2-2::LUC is active in all plant tissues, but is strongly modulated in cotyledons upon germination, in leaves in response to desiccation, cold treatment, wounding, or ultraviolet-B light, and in petal tissue in response to pollination of the stigma. Analysis of mRNA levels indicated that the modulations in ZPT2-2::LUC expression reflect modulations in endogenous ZPT2-2 gene expression. The change in ZPT2-2::LUC activity by cold treatment, wounding, desiccation, and ultraviolet-B light suggest that the phytohormones ethylene and jasmonic acid are involved in regulating the expression of ZPT2-2. Although up-regulation of expression of ZPT2-2 can be blocked by inhibitors of ethylene perception, expression in plants is not induced by exogenously applied ethylene. The application of jasmonic acid does result in an up-regulation of gene activity and, thus, ZPT2-2 may play a role in the realization of the jasmonic acid hormonal responses in petunia. PMID:10594102

Making Sense of Missense in the Lynch Syndrome: The Clinical Perspective

PubMed Central

Lynch, Henry T.; Jascur, Thomas; Lanspa, Stephen; Boland, C. Richard

2010-01-01

The DNA mismatch repair system provides critical genetic housekeeping, and its failure is associated with tumorigenesis. Through distinct domains on the DNA mismatch repair proteins, the system recognizes and repairs errors occurring during DNA synthesis, but signals apoptosis when the DNA damage cannot be repaired. Certain missense mutations in the mismatch repair genes can selectively alter just one of these functions. This impacts the clinical features of tumors associated with defective DNA mismatch repair activity. New work reported by Xie et al. in this issue of the journal (beginning on page XXX) adds to the understanding of DNA mismatch repair. PMID:20978117

Chromosomal instability in women with primary ovarian insufficiency.

PubMed

Katari, Sunita; Aarabi, Mahmoud; Kintigh, Angela; Mann, Susan; Yatsenko, Svetlana A; Sanfilippo, Joseph S; Zeleznik, Anthony J; Rajkovic, Aleksandar

2018-02-07

What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome damage, may not be representative of all the affected tissues. Another limitation pertains to the MMC assay which detects homologous repair pathway defects and does not test deficiencies in other DNA repair pathways. Our results provide evidence for functional impairment of DNA repair in idiopathic POI, which may predispose the patients to other DNA repair-related conditions such as accelerated aging and/or cancer susceptibility. Funding was provided by the National Institute of Child Health and Human Development. There were no competing interests to declare. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

PubMed Central

Cohen-Tannoudji, Michel; Robine, Sylvie; Choulika, André; Pinto, Daniel; El Marjou, Fatima; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric

1998-01-01

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest. PMID:9488460

Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.

PubMed

Molina-Estevez, F Javier; Nowrouzi, Ali; Lozano, M Luz; Galy, Anne; Charrier, Sabine; von Kalle, Christof; Guenechea, Guillermo; Bueren, Juan A; Schmidt, Manfred

2015-01-01

Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.

Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

PubMed

Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Aspesi, Anna; Morleo, Giulia; Biasi, Alessandra; Sculco, Marika; Mancuso, Giuseppe; Guarrera, Simonetta; Righi, Luisella; Grosso, Federica; Libener, Roberta; Pavesi, Mansueto; Mariani, Narciso; Casadio, Caterina; Boldorini, Renzo; Mirabelli, Dario; Pasini, Barbara; Magnani, Corrado; Matullo, Giuseppe; Dianzani, Irma

2017-10-01

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Inactivation of antibiotic resistance genes in municipal wastewater by chlorination, ultraviolet, and ozonation disinfection.

PubMed

Zhuang, Yao; Ren, Hongqiang; Geng, Jinju; Zhang, Yingying; Zhang, Yan; Ding, Lili; Xu, Ke

2015-05-01

This study investigated the inactivation of two antibiotic resistance genes (ARGs)-sul1 and tetG, and the integrase gene of class 1 integrons-intI1 by chlorination, ultraviolet (UV), and ozonation disinfection. Inactivation of sul1, tetG, and intI1 underwent increased doses of three disinfectors, and chlorine disinfection achieved more inactivation of ARGs and intI1 genes (chlorine dose of 160 mg/L with contact time of 120 min for 2.98-3.24 log reductions of ARGs) than UV irradiation (UV dose of 12,477 mJ/cm(2) for 2.48-2.74 log reductions of ARGs) and ozonation disinfection (ozonation dose of 177.6 mg/L for 1.68-2.55 log reductions of ARGs). The 16S rDNA was more efficiently removed than ARGs by ozone disinfection. The relative abundance of selected genes (normalized to 16S rDNA) increased during ozonation and with low doses of UV and chlorine disinfection. Inactivation of sul1 and tetG showed strong positive correlations with the inactivation of intI1 genes (for sul1, R (2)  = 0.929 with p < 0.01; for tetG, R (2)  = 0.885 with p < 0.01). Compared to other technologies (ultraviolet disinfection, ozonation disinfection, Fenton oxidation, and coagulation), chlorination is an alternative method to remove ARGs from wastewater effluents. At a chlorine dose of 40 mg/L with 60 min contact time, the selected genes inactivation efficiency could reach 1.65-2.28 log, and the cost was estimated at 0.041 yuan/m(3).

Genetic polymorphisms in 19q13.3 genes associated with alteration of repair capacity to BPDE-DNA adducts in primary cultured lymphocytes.

PubMed

Xiao, Mingyang; Xiao, Sha; Straaten, Tahar van der; Xue, Ping; Zhang, Guopei; Zheng, Xiao; Zhang, Qianye; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Zhu, Guolian; Lu, Xiaobo

2016-12-01

Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage in cells caused by BPDE is normally repaired by Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Genetic variations in NER and BER can change individual DNA repair capacity to DNA damage induced by BPDE. In the present study we determined the number of in vitro induced BPDE-DNA adducts in lymphocytes, to reflect individual susceptibility to Polycyclic aromatic hydrocarbons (PAHs)-induced carcinogenesis. The BPDE-DNA adduct level in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 281 randomly selected participants. We genotyped for 9 single nucleotide polymorphisms (SNPs) in genes involved in NER (XPB rs4150441, XPC rs2228001, rs2279017 and XPF rs4781560), BER (XRCC1 rs25487, rs25489 and rs1799782) and genes located on chromosome 19q13.2-3 (PPP1R13L rs1005165 and CAST rs967591). We found that 3 polymorphisms in chromosome 19q13.2-3 were associated with lower levels of BPDE-DNA adducts (MinorT allele in XRCC1 rs1799782, minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571). In addition, a modified comet assay was performed to further confirm the above conclusions. We found both minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571 were associated with the lower levels of BPDE-adducts. Our data suggested that the variant genotypes of genes in chromosome 19q13.2-3 are associated with the alteration of repair efficiency to DNA damage caused by Benzo[a]pyrene, and may contribute to enhance predictive value for individual's DNA repair capacity in response to environmental carcinogens. Copyright © 2016 Elsevier B.V. All rights reserved.

The repair of low dose UV light-induced damage to human skin DNA in condition of trace amount Mg 2+

NASA Astrophysics Data System (ADS)

Gao, Fang; Guo, Zhouyi; Zheng, Changchun; Wang, Rui; Liu, Zhiming; Meng, Pei; Zhai, Juan

2008-12-01

Ultraviolet light-induced damage to human skin DNA was widely investigated. The primary mechanism of this damage contributed to form cyclobutane pyrimidine dimmers (CPDs). Although the distribution of UV light-induced CPDs within a defined sequence is similar, the damage in cellular environment which shields the nuclear DNA was higher than that in organism in apparent dose. So we use low UVB light as main study agent. Low dose UV-irradiated HDF-a cells (Human Dermal Fibroblasts-adult cells) which is weaker than epidermic cells were cultured with DMEM at different trace amount of Mg2+ (0mmol/L , 0.1mmol/L , 0.2mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L) free-serum DMEM and the repair of DNA strands injured were observed. Treat these cells with DNA strand breaks detection, photoproducts detection and the repair of photoproducts detection. Then quantitate the role of trace amount Mg2+ in repair of UV light-induced damage to human skin. The experiment results indicated that epidermic cells have capability of resistance to UV-radiation at a certain extent. And Mg2+ can regulate the UV-induced damage repair and relative vitality. It can offer a rationale and experiment data to relieve UV light-induced skin disease.

[Light protection for xeroderma pigmentosum].

PubMed

Ettinger, M; Berneburg, M

2017-05-01

Xeroderma pigmentosum is a rare autosomal recessive disorder which is caused by germinal mutations responsible for the repair of ultraviolet (UV) radiation-induced DNA lesions. It is characterized by hypersensitivity to UV radiation, poikiloderma, ocular surface disease, and in some patients pronounced sunburn and neurological disease. Patients have a very high risk of developing ocular and skin cancer on exposed body sites. No cure is available for these patients except complete protection from all types of UV radiation.

Vitamin E Modifies High-Fat Diet-Induced Increase of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice.

PubMed

Remely, Marlene; Ferk, Franziska; Sterneder, Sonja; Setayesh, Tahereh; Kepcija, Tatjana; Roth, Sylvia; Noorizadeh, Rahil; Greunz, Martina; Rebhan, Irene; Wagner, Karl-Heinz; Knasmüller, Siegfried; Haslberger, Alexander

2017-06-14

Obesity is associated with low-grade inflammation, increased ROS production and DNA damage. Supplementation with antioxidants might ameliorate DNA damage and support epigenetic regulation of DNA repair. C57BL/6J male mice were fed a high-fat (HFD) or a control diet (CD) with and without vitamin E supplementation (4.5 mg/kg body weight (b.w.)) for four months. DNA damage, DNA promoter methylation and gene expression of Dnmt1 and a DNA repair gene ( MLH1 ) were assayed in liver and colon. The HFD resulted in organ specific changes in DNA damage, the epigenetically important Dnmt1 gene, and the DNA repair gene MLH1 . Vitamin E reduced DNA damage and showed organ-specific effects on MLH1 and Dnmt1 gene expression and methylation. These results suggest that interventions with antioxidants and epigenetic active food ingredients should be developed as an effective prevention for obesity-and oxidative stress-induced health risks.

Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

PubMed Central

Mikhed, Yuliya; Görlach, Agnes; Knaus, Ulla G.; Daiber, Andreas

2015-01-01

Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease. PMID:26079210

Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells.

PubMed

Lerner, Leticia K; Francisco, Guilherme; Soltys, Daniela T; Rocha, Clarissa R R; Quinet, Annabel; Vessoni, Alexandre T; Castro, Ligia P; David, Taynah I P; Bustos, Silvina O; Strauss, Bryan E; Gottifredi, Vanesa; Stary, Anne; Sarasin, Alain; Chammas, Roger; Menck, Carlos F M

2017-02-17

Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

EUV multilayer defect compensation (MDC) by absorber pattern modification: from theory to wafer validation

NASA Astrophysics Data System (ADS)

Pang, Linyong; Hu, Peter; Satake, Masaki; Tolani, Vikram; Peng, Danping; Li, Ying; Chen, Dongxue

2011-11-01

According to the ITRS roadmap, mask defects are among the top technical challenges to introduce extreme ultraviolet (EUV) lithography into production. Making a multilayer defect-free extreme ultraviolet (EUV) blank is not possible today, and is unlikely to happen in the next few years. This means that EUV must work with multilayer defects present on the mask. The method proposed by Luminescent is to compensate effects of multilayer defects on images by modifying the absorber patterns. The effect of a multilayer defect is to distort the images of adjacent absorber patterns. Although the defect cannot be repaired, the images may be restored to their desired targets by changing the absorber patterns. This method was first introduced in our paper at BACUS 2010, which described a simple pixel-based compensation algorithm using a fast multilayer model. The fast model made it possible to complete the compensation calculations in seconds, instead of days or weeks required for rigorous Finite Domain Time Difference (FDTD) simulations. Our SPIE 2011 paper introduced an advanced compensation algorithm using the Level Set Method for 2D absorber patterns. In this paper the method is extended to consider process window, and allow repair tool constraints, such as permitting etching but not deposition. The multilayer defect growth model is also enhanced so that the multilayer defect can be "inverted", or recovered from the top layer profile using a calibrated model.

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer

PubMed Central

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2014-01-01

Purpose The mismatch repair (MMR) system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in the MMR proteins have a 102 –103-fold increase in the mutation rate. Single nucleotide polymorphisms (SNPs) of MMR genes have been shown to cause a reduction in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer (PC) and that p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. Material and Methods DNA samples from 110 cases of prostate cancer and healthy controls (n=110) were analyzed by SSCP and PCR-RFLP to determine the genotypic frequency of five different polymorphic loci on two MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare the genotype frequency between patients and controls. Results A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR, 1.87; 95% CI, 1.0–3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR, 1.57; 95% CI, 0.92–2.72). Among p53 codon72 Arg/Pro + Pro/Pro carriers, the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR = 2.1, 95% CI; 1.05–4.34). Conclusion This is the first report on the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer. PMID:18355840

Mismatch repair polymorphisms and the risk of colorectal cancer.

PubMed

Berndt, Sonja I; Platz, Elizabeth A; Fallin, M Daniele; Thuita, Lucy W; Hoffman, Sandra C; Helzlsouer, Kathy J

2007-04-01

Rare germline variants in mismatch repair genes have been linked to hereditary nonpolyposis colorectal cancer; however, it is unknown whether common polymorphisms in these genes alter the risk of colorectal cancer. To examine the association between common variants in mismatch repair genes and colorectal cancer, we conducted a case-cohort study within the CLUE II cohort. Four single nucleotide polymorphisms in 3 mismatch repair genes (MSH3 R940Q, MSH3 T1036A, MSH6 G39E and MLH1 I219V) were genotyped in 237 colorectal cancer cases and a subcohort of 2,189 participants. Incidence rate ratios (RRs) and 95% confidence intervals (95% CIs) for each polymorphism were estimated. The MSH3 1036A variant was found to be associated with an increased risk of colorectal cancer (RR=1.28, 95% CI: 0.94-1.74 and RR=1.65, 95% CI: 1.01-2.70 for the AT and TT genotypes, respectively, with p(trend)=0.02), particularly proximal colon cancer. Although the MSH3 940Q variant was only weakly associated with colorectal cancer overall (p(trend)=0.07), it was associated with a significant increased risk of proximal colon cancer (RR=1.69, 95% CI: 1.10-2.61 and RR=2.68, 95% CI: 0.96-7.47 for the RQ and QQ genotypes, respectively with p(trend)=0.005). Processed meat intake appeared to modify the association between the MSH3 polymorphisms and colorectal cancer (p(interaction) < 0.10 for both). No association was observed with the MSH6 and MLH1 polymorphisms overall. This study suggests that common polymorphisms in the mismatch repair gene, MSH3, may increase the risk of colorectal cancer, especially proximal colon cancer. (c) 2006 Wiley-Liss, Inc.

Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

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

Vermeulen, W.; Kleijer, W.J.; Bootsma, D.

1994-02-01

The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with the remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in themore » vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here the authors report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of >40 years. Analysis of the frequency of hprt[sup [minus

Clinical potential and challenges of using genetically modified cells for articular cartilage repair.

PubMed

Madry, Henning; Cucchiarini, Magali

2011-06-01

Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

PubMed

Guo, Xiaoge; Jinks-Robertson, Sue

2013-12-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations. Copyright © 2013 Elsevier B.V. All rights reserved.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

PubMed Central

Guo, Xiaoge; Jinks-Robertson, Sue

2013-01-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral introduction of UV damage can potentially compromise genetic interpretations. PMID:24120148

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light

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

Cleaver, J.E.

1989-01-01

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were notmore » significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.« less

An Exercise to Estimate Differential Gene Expression in Human Cells

ERIC Educational Resources Information Center

Chaudhry, M. Ahmad

2006-01-01

The expression of genes in cells of various tissue types varies considerably and is correlated with the function of a particular organ. The pattern of gene expression changes in diseased tissues, in response to therapy or infection and exposure to environmental mutagens, chemicals, ultraviolet light, and ionizing radiation. To better understand…

Identification of novel target genes involved in Indian Fanconi anemia patients using microarray.

PubMed

Shyamsunder, Pavithra; Ganesh, Kripa S; Vidyasekar, Prasanna; Mohan, Sheila; Verma, Rama Shanker

2013-12-01

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype. © 2013 Elsevier B.V. All rights reserved.

X-ray irradiation has positive effects for the recovery of peripheral nerve injury maybe through the vascular smooth muscle contraction signaling pathway.

PubMed

Jiang, Bo; Zhang, Yong; She, Chang; Zhao, Jiaju; Zhou, Kailong; Zuo, Zhicheng; Zhou, Xiaozhong; Wang, Peiji; Dong, Qirong

2017-09-01

It is well known that moderate to high doses of ionizing radiation have a toxic effect on the organism. However, there are few experimental studies on the mechanisms of LDR ionizing radiation on nerve regeneration after peripheral nerve injury. We established the rats' peripheral nerve injury model via repaired Peripheral nerve injury nerve, vascular endothelial growth factor a and Growth associated protein-43 were detected from different treatment groups. We performed transcriptome sequencing focusing on investigating the differentially expressed genes and gene functions between the control group and 1Gy group. Sequencing was done by using high-throughput RNA-sequencing (RNA-seq) technologies. The results showed the 1Gy group to be the most effective promoting repair. RNA-sequencing identified 619 differently expressed genes between control and treated groups. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways. Among them, candidate genes associated with nerve repair were identified. Pathways involved in cell-substrate adhesion, vascular smooth muscle contraction and cell adhesion molecule signaling may be involved in recovery from peripheral nerve injury. Copyright © 2017. Published by Elsevier B.V.

Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development.

PubMed

Zhu, Yan; Weng, Minjie; Yang, Yue; Zhang, Chi; Li, Ziyu; Shen, Wen-Hui; Dong, Aiwu

2011-05-01

Anti-silencing function1 (ASF1) is an evolutionarily conserved histone chaperone. Studies in yeast and animals indicate that ASF1 proteins play important roles in various chromatin-based processes, including gene transcription, DNA replication and repair. While two genes encoding ASF1 homologues, AtASF1A and AtASF1B, are found in the Arabidopsis genome, their function has not been studied. Here we report that both AtASF1A and AtASF1B proteins bind histone H3, and are localized in the cytoplasm and the nucleus. Loss-of-function of either AtASF1A or AtASF1B did not show obvious defects, whereas simultaneous knockdown of both genes in the double mutant Atasf1ab drastically inhibited plant growth and caused abnormal vegetative and reproductive organ development. The Atasf1ab mutant plants exhibit cell number reduction, S-phase delay/arrest, and reduced polyploidy levels. Selective up-regulation of expression of a subset of genes, including those involved in S-phase checkpoints and the CYCB1;1 gene at the G₂-to-M transition, was observed in Atasf1ab. Furthermore, the Atasf1ab-triggered replication fork stalling constitutively activates the DNA damage checkpoint and repair genes, including ATM, ATR, PARP1 and PARP2 as well as several genes of the homologous recombination (HR) pathway but not genes of the non-homologous end joining (NHEJ) pathway. In spite of the activation of repair genes, an increased level of DNA damage was detected in Atasf1ab, suggesting that defects in the mutant largely exceed the available capacity of the repair machinery. Taken together, our study establishes crucial roles for the AtASF1A and AtASF1B genes in chromatin replication, maintenance of genome integrity and cell proliferation during plant development. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Regulation of SNM1, an inducible Saccharomyces cerevisiae gene required for repair of DNA cross-links.

PubMed

Wolter, R; Siede, W; Brendel, M

1996-02-05

The interstrand cross-link repair gene SNM1 of Saccharomyces cerevisiae was examined for regulation in response to DNA-damaging agents. Induction of SNM1-lacZ fusions was detected in response to nitrogen mustard, cis-platinum (II) diamine dichloride, UV light, and 8-methoxypsoralen + UVA, but not after heat-shock treatment or incubation with 2-dimethylaminoethylchloride, methylmethane sulfonate or 4-nitroquinoline-N-oxide. The promoter of SNM1 contains a 15 bp motif, which shows homology to the DRE2 box of the RAD2 promoter. Similar motifs have been found in promoter regions of other damage-inducible DNA repair genes. Deletion of this motif results in loss of inducibility of SNM1. Also, a putative negative upstream regulation sequence was found to be responsible for repression of constitutive transcription of SNM1. Surprisingly, no inducibility of SNM1 was found after treatment with DNA-damaging agents in strains without an intact DUN1 gene, while regulation seems unchanged in sad1 mutants.

Fanconi anemia: a disorder defective in the DNA damage response.

PubMed

Kitao, Hiroyuki; Takata, Minoru

2011-04-01

Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

Polymorphic Variation in Double Strand Break Repair Gene in Indian Population: A Comparative Approach with Worldwide Ethnic Group Variations.

PubMed

Mandal, Raju Kumar; Mittal, Rama Devi

2018-04-01

DNA repair capacity is essential in maintaining cellular functions and homeostasis. Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. Double strand break repair pathway plays critical roles in maintaining genome stability. Present study was conducted to determine distribution of XRCC3 Exon 7 (C18067T, rs861539) and XRCC7 Intron 8 (G6721T, rs7003908) gene polymorphisms in North Indian population and compare with different populations globally. The genotype assays were performed in 224 normal healthy individuals of similar ethnicity using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Allelic frequencies of wild type were 79% (C) in XRCC3 Exon 7 C > T and 57% (G) in XRCC7 Intron 8 (G > T) 57% (G) observed. On the other hand, the variant allele frequency were 21% (T) in XRCC3 Exon 7 C > T and 43% (T) in XRCC7 Intron 8 G > T respectively. Major differences from other ethnic populations were observed. Our results suggest that frequency in these DNA repair genes exhibit distinctive pattern in India that could be attributed to ethnicity variation. This could assist in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

PubMed

O'Hanlon, Karen A; Margison, Geoffrey P; Hatch, Amy; Fitzpatrick, David A; Owens, Rebecca A; Doyle, Sean; Jones, Gary W

2012-09-01

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus

PubMed Central

O’Hanlon, Karen A.; Margison, Geoffrey P.; Hatch, Amy; Fitzpatrick, David A.; Owens, Rebecca A.; Doyle, Sean; Jones, Gary W.

2012-01-01

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O6-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O6-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system. PMID:22669901

Glial kon/NG2 gene network for central nervous system repair.

PubMed

Losada-Perez, Maria; Harrison, Neale; Hidalgo, Alicia

2017-01-01

The glial regenerative response to central nervous system (CNS) injury, although limited, can be harnessed to promote regeneration and repair. Injury provokes the proliferation of ensheathing glial cells, which can differentiate to remyelinate axons, and partially restore function. This response is evolutionarily conserved, strongly implying an underlying genetic mechanism. In mammals, it is elicited by NG2 glia, but most often newly generated cells fail to differentiate. Thus an important goal had been to find out how to promote glial differentiation following the proliferative response. A gene network involving Notch and prospero (pros) controls the balance between glial proliferation and differentiation in flies and mice, and promotes CNS repair at least in fruit-flies. A key missing link had been how to relate the function of NG2 to this gene network. Recent findings by Losada-Perez et al., published in JCB, demonstrated that the Drosophila NG2 homologue kon-tiki (kon) is functionally linked to Notch and pros in glia. By engaging in two feedback loops with Notch and Pros, in response to injury, Kon can regulate both glial cell number and glial shape homeostasis, essential for repair. Drosophila offers powerful genetics to unravel the control of stem and progenitor cells for regeneration and repair.

Emergence of a daptomycin-non-susceptible Enterococcus faecium strain that encodes mutations in DNA repair genes after high-dose daptomycin therapy.

PubMed

Matono, Takashi; Hayakawa, Kayoko; Hirai, Risen; Tanimura, Akira; Yamamoto, Kei; Fujiya, Yoshihiro; Mawatari, Momoko; Kutsuna, Satoshi; Takeshita, Nozomi; Mezaki, Kazuhisa; Ohmagari, Norio; Miyoshi-Akiyama, Tohru

2016-04-01

An increasing number of reports have documented the emergence of daptomycin-nonsusceptible Enterococcus in patients during daptomycin therapy. Even though several mechanisms for daptomycin-nonsusceptibility have been suggested, the potential genetic mutations which might contribute to the daptomycin-nonsusceptibility are not fully understood. We isolated a vancomycin-susceptible, daptomycin nonsusceptible Enterococcus faecium strain from a patient with acute lymphocytic leukemia who received high-dose daptomycin therapy for E. faecium endocarditis. Whole-genome sequencing analysis revealed mutations within genes encoding DNA repair proteins MutL and RecJ of the daptomycin-nonsusceptible Enterococcus strain which might have facilitated its emergence. We identified the mutations of DNA mismatch repair genes in a clinical isolate of daptomycin nonsusceptible E. faecium which emerged in spite of high-dose daptomycin therapy. The finding implicates the possible association of DNA repair mechanism and daptomycin resistance. Careful monitoring is necessary to avoid the emergence of daptomycin non-susceptible isolates of E. faecium and particularly in cases of long-term daptomycin use or in immunocompromised patients.

Association of Leukotrichia in Vitiligo and Asp148Glu Polymorphism of Apurinic/Apyrimidinic Endonuclease 1.

PubMed

Aydin, A Fatih; Aydıngöz, İkbal Esen; Doğru-Abbasoğlu, Semra; Vural, Pervin; Uysal, Müjdat

2017-01-01

Oxidative stress and increased DNA damage have been implicated in the etiopathogenesis of vitiligo. Oxidative DNA damage is mainly repaired by the base excision repair (BER) pathway. We sought to determine whether polymorphisms in DNA repair genes may have a role in the pathogenesis of vitiligo. We conducted a study including 100 patients with vitiligo and age- and sex-matched 193 control subjects to examine the role of single-nucleotide polymorphisms of BER genes, human 8-oxoG DNA N-glycosylase 1 (codon 326), apurinic/apyrimidinic endonuclease 1 (APE1) (codon 148), and X-ray repair cross-complementing group 1 (codon 399) as risk factors for vitiligo. These polymorphisms were determined by quantitative real-time polymerase chain reaction and melting curve analysis. No significant association was observed between the variant alleles of studied genes and vitiligo. However, we showed that the presence of APE1 148Glu variant allele is associated with leukotrichia. This preliminary study suggests that APE1 (codon 148) polymorphism may play a role in vitiligo pathogenesis.

Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

Cancer.gov

Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have been associated with DSBs, including modifications of histone tails and exchange of histone variants, some increasing chromatin accessibility, others reducing it. In fact, distinct domains flanking a single DSB have been observed that are bound by opposing repair pathway proteins 53BP1and BRCA1, which promote non-homologous end joining (NHEJ) and homologous recombination (HR), respectively. To investigate whether DSB-proximal chromatin reorganization affects repair pathway selection, Philipp Oberdoerffer, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues performed a high-throughput RNA interference (RNAi) screen for chromatin-related genes that modulate HR.

DIMETHYLARSINIC ACID ALTERS EXPRESSION OF OXIDATIVE STRESS AND DNA REPAIR GENES IN A DOSE DEPENDENT MANNER IN THE TRANSITIONAL EPITHELIUM OF THE URINARY BLADDER FROM FEMALE F344 RATS.

EPA Science Inventory

Dose-dependent alteration of oxidative stress and DNA repair gene expression by Dimethylarsinic acid [DMA(V)] in transitional epithelium of urinary bladder from female F344 rats.
Arsenic (As) is a major concern as millions of people are at risk from drinking arsenic contaminat...

8-Oxoguanine DNA glycosylase1-driven DNA repair-A paradoxical role in lung aging.

PubMed

German, Peter; Saenz, David; Szaniszlo, Peter; Aguilera-Aguirre, Leopoldo; Pan, Lang; Hegde, Muralidhar L; Bacsi, Attila; Hajas, Gyorgy; Radak, Zsolt; Ba, Xueqing; Mitra, Sankar; Papaconstantinou, John; Boldogh, Istvan

2017-01-01

Age-associated changes in lung structure and function are some of the most important predictors of overall health, cognitive activities and longevity. Common to all aging cells is an increase in oxidatively modified DNA bases, primarily 8-oxo-7,8-dihydroguanine (8-oxoG). It is repaired via DNA base excision repair pathway driven by 8-oxoguanine DNA glycosylase-1 (OGG1-BER), whose role in aging has been the focus of many studies. This study hypothesizes that signaling and consequent gene expression during cellular response to OGG1-BER "wires" senescence/aging processes. To test OGG1-BER was mimicked by repeatedly exposing diploid lung fibroblasts cells and airways of mice to 8-oxoG base. Results showed that repeated exposures led to G1 cell cycle arrest and pre-matured senescence of cultured cells in which over 1000 genes were differentially expressed -86% of them been identical to those in naturally senesced cells. Gene ontology analysis of gene expression displayed biological processes driven by small GTPases, phosphoinositide 3-kinase and mitogen activated kinase cascades both in cultured cells and lungs. These results together, points to a new paradigm about the role of DNA damage and repair by OGG1 in aging and age-associated disease processes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

PubMed Central

Rattray, A J; Shafer, B K; Garfinkel, D J

2000-01-01

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA. PMID:10655210

PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

PubMed

Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

2017-03-27

Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

PubMed Central

Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

2013-01-01

After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity.

PubMed

Boglev, Yeliz; Wilanowski, Tomasz; Caddy, Jacinta; Parekh, Vishwas; Auden, Alana; Darido, Charbel; Hislop, Nikki R; Cangkrama, Michael; Ting, Stephen B; Jane, Stephen M

2011-01-15

The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development. Copyright © 2010 Elsevier Inc. All rights reserved.

Cell cycle-related genes as modifiers of age of onset of colorectal cancer in Lynch syndrome: a large-scale study in non-Hispanic white patients.

PubMed

Chen, Jinyun; Pande, Mala; Huang, Yu-Jing; Wei, Chongjuan; Amos, Christopher I; Talseth-Palmer, Bente A; Meldrum, Cliff J; Chen, Wei V; Gorlov, Ivan P; Lynch, Patrick M; Scott, Rodney J; Frazier, Marsha L

2013-02-01

Heterogeneity in age of onset of colorectal cancer in individuals with mutations in DNA mismatch repair genes (Lynch syndrome) suggests the influence of other lifestyle and genetic modifiers. We hypothesized that genes regulating the cell cycle influence the observed heterogeneity as cell cycle-related genes respond to DNA damage by arresting the cell cycle to provide time for repair and induce transcription of genes that facilitate repair. We examined the association of 1456 single nucleotide polymorphisms (SNPs) in 128 cell cycle-related genes and 31 DNA repair-related genes in 485 non-Hispanic white participants with Lynch syndrome to determine whether there are SNPs associated with age of onset of colorectal cancer. Genotyping was performed on an Illumina GoldenGate platform, and data were analyzed using Kaplan-Meier survival analysis, Cox regression analysis and classification and regression tree (CART) methods. Ten SNPs were independently significant in a multivariable Cox proportional hazards regression model after correcting for multiple comparisons (P < 5 × 10(-4)). Furthermore, risk modeling using CART analysis defined combinations of genotypes for these SNPs with which subjects could be classified into low-risk, moderate-risk and high-risk groups that had median ages of colorectal cancer onset of 63, 50 and 42 years, respectively. The age-associated risk of colorectal cancer in the high-risk group was more than four times the risk in the low-risk group (hazard ratio = 4.67, 95% CI = 3.16-6.92). The additional genetic markers identified may help in refining risk groups for more tailored screening and follow-up of non-Hispanic white patients with Lynch syndrome.

Gene expression analysis upon lncRNA DDSR1 knockdown in human fibroblasts

PubMed Central

Jia, Li; Sun, Zhonghe; Wu, Xiaolin; Misteli, Tom; Sharma, Vivek

2015-01-01

Long non-coding RNAs (lncRNAs) play important roles in regulating diverse biological processes including DNA damage and repair. We have recently reported that the DNA damage inducible lncRNA DNA damage-sensitive RNA1 (DDSR1) regulates DNA repair by homologous recombination (HR). Since lncRNAs also modulate gene expression, we identified gene expression changes upon DDSR1 knockdown in human fibroblast cells. Gene expression analysis after RNAi treatment targeted against DDSR1 revealed 119 genes that show differential expression. Here we provide a detailed description of the microarray data (NCBI GEO accession number GSE67048) and the data analysis procedure associated with the publication by Sharma et al., 2015 in EMBO Reports [1]. PMID:26697398

Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes

PubMed Central

Mallet, Justin D.; Bastien, Nathalie; Gendron, Sébastien P.; Rochette, Patrick J.

2016-01-01

Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

USGS Publications Warehouse

Kienzler, Aude; Mahler, Barbara J.; Van Metre, Peter C.; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-01-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line.

PubMed

Kienzler, Aude; Mahler, Barbara J; Van Metre, Peter C; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-07-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Repair of astrocytes, blood vessels, and myelin in the injured brain: possible roles of blood monocytes

PubMed Central

2013-01-01

Inflammation in injured tissue has both repair functions and cytotoxic consequences. However, the issue of whether brain inflammation has a repair function has received little attention. Previously, we demonstrated monocyte infiltration and death of neurons and resident microglia in LPS-injected brains (Glia. 2007. 55:1577; Glia. 2008. 56:1039). Here, we found that astrocytes, oligodendrocytes, myelin, and endothelial cells disappeared in the damage core within 1–3 d and then re-appeared at 7–14 d, providing evidence of repair of the brain microenvironment. Since round Iba-1+/CD45+ monocytes infiltrated before the repair, we examined whether these cells were involved in the repair process. Analysis of mRNA expression profiles showed significant upregulation of repair/resolution-related genes, whereas proinflammatory-related genes were barely detectable at 3 d, a time when monocytes filled injury sites. Moreover, Iba-1+/CD45+ cells highly expressed phagocytic activity markers (e.g., the mannose receptors, CD68 and LAMP2), but not proinflammatory mediators (e.g., iNOS and IL1β). In addition, the distribution of round Iba-1+/CD45+ cells was spatially and temporally correlated with astrocyte recovery. We further found that monocytes in culture attracted astrocytes by releasing soluble factor(s). Together, these results suggest that brain inflammation mediated by monocytes functions to repair the microenvironment of the injured brain. PMID:23758980

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

PubMed Central

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

1994-01-01

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

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress.

PubMed

Lee, Ming-Hsiang; Wang, Liya; Chang, Zee-Fen

2014-04-01

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.

Blocking by the carcinogen, L-ethionine, of SOS functions in a tif-1 mutant of Escherichia coli B/r.

PubMed

Wiesner, R; Troll, W

1981-11-01

In Escherichia coli, DNA damage by carcinogenic agents results in the coordinate expression of a diversity of functions (SOS functions), many of which are thermally inducible without any damage to DNA in a tif-1 mutant. These include prophage induction, filamentous growth, and an error-prone DNA repair activity, which is responsible for ultraviolet-induced mutagenesis. Ethionine causes hepatic carcinoma in rats after prolonged feeding but is not a mutagen in the Ames test. The present study shows that 10 mM ethionine prevents the thermal induction of lambda-prophage in a tif-1 derivative of E. coli. The enhancement of mutation, which normally occurs at high temperature after a low dose of ultraviolet light, is also blocked by ethionine. Ethionine does not block, to any appreciable extent, the incorporation of radioactive precursors into RNA, DNA, or protein.

Interactive lethal and mutagenic effects of ultraviolet light and bleomycin in yeast: synergism or antagonism?

PubMed

Lillo, O L; Severgnini, A A; Nunes, E M

1997-11-01

The mutagenic interactions of ultraviolet light and bleomycin in haploid populations of Saccharomyces cerevisiae were analyzed. Survival and mutation frequency as a function of different bleomycin concentrations after one conditioning dose of UV radiation were determined. Furthermore, corresponding interaction functions and sensitization factors were calculated. A synergistic interaction between UV light and bleomycin was shown for both lethal and mutagenic events when the cells were in nutrient broth during the treatments. Conversely, the interaction between UV light and bleomycin was antagonistic when the cells were in deionized water during the treatment. The magnitude of lethal and mutagenic interactions depends on dose, and thus presumably on the number of lesions. The observed interactions between UV light and bleomycin suggest that the mechanism that is most likely involved is the induction of repair systems with different error probabilities during the delay of cell division.

The Effect of Polymorphisms in DNA Repair Genes and Carcinogen Metabolizers on Leukocyte Telomere Length: A Cohort of Healthy Spanish Smokers.

PubMed

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

2016-04-01

Smoking implies exposure to carcinogenic agents that causes DNA damage, which could be suspected to enhance telomere attrition. To protect and deal with DNA damage, cells possess mechanisms that repair and neutralize harmful substances. Polymorphisms altering DNA repair capacity or carcinogen metabolism may lead to synergistic effects with tobacco carcinogen-induced shorter telomere length independently of cancer interaction. The aim of this study was to explore the association between leukocyte telomere length (LTL) and several genetic polymorphisms in DNA repair genes and carcinogen metabolizers in a cohort of healthy smokers. We evaluated the effect of six genetic polymorphisms in cytochrome P1A1 (Ile462Val), XRCC1 (Arg399Gln), APEX1 (Asp148Glu), XRCC3 (Thr241Met), and XPD (Asp312Asn; Lys751Gln) on LTL in a cohort of 145 healthy smokers in addition to smoking habits. Logistic regression analysis showed an association between XRCC1 399Gln allele and shorter telomere length (OR = 5.03, 95% CI = 1.08% to 23.36%). There were not association between the rest of polymorphisms analyzed and LTL. Continuous exposure to tobacco could overwhelm the DNA repair machinery, making the effect of the polymorphisms that reduce repair capacity more pronounced. Analyzing the function of smoking-induced DNA-repair genes and LTL is an important goal in order to identify therapeutic targets to treat smoking-induced diseases. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.

PubMed

Liang, Puping; Xu, Yanwen; Zhang, Xiya; Ding, Chenhui; Huang, Rui; Zhang, Zhen; Lv, Jie; Xie, Xiaowei; Chen, Yuxi; Li, Yujing; Sun, Ying; Bai, Yaofu; Songyang, Zhou; Ma, Wenbin; Zhou, Canquan; Huang, Junjiu

2015-05-01

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

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

PubMed Central

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

1996-01-01

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

Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress.

PubMed

Kristensen, Ulrik; Epanchintsev, Alexey; Rauschendorf, Marc-Alexander; Laugel, Vincent; Stevnsner, Tinna; Bohr, Vilhelm A; Coin, Frédéric; Egly, Jean-Marc

2013-06-18

Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.

Genomic Approach to Understand the Association of DNA Repair with Longevity and Healthy Aging Using Genomic Databases of Oldest-Old Population

PubMed Central

Kim, Hyun Soo

2018-01-01

Aged population is increasing worldwide due to the aging process that is inevitable. Accordingly, longevity and healthy aging have been spotlighted to promote social contribution of aged population. Many studies in the past few decades have reported the process of aging and longevity, emphasizing the importance of maintaining genomic stability in exceptionally long-lived population. Underlying reason of longevity remains unclear due to its complexity involving multiple factors. With advances in sequencing technology and human genome-associated approaches, studies based on population-based genomic studies are increasing. In this review, we summarize recent longevity and healthy aging studies of human population focusing on DNA repair as a major factor in maintaining genome integrity. To keep pace with recent growth in genomic research, aging- and longevity-associated genomic databases are also briefly introduced. To suggest novel approaches to investigate longevity-associated genetic variants related to DNA repair using genomic databases, gene set analysis was conducted, focusing on DNA repair- and longevity-associated genes. Their biological networks were additionally analyzed to grasp major factors containing genetic variants of human longevity and healthy aging in DNA repair mechanisms. In summary, this review emphasizes DNA repair activity in human longevity and suggests approach to conduct DNA repair-associated genomic study on human healthy aging.

Microorganisms and biomolecules in space hard environment

NASA Technical Reports Server (NTRS)

Horneck, G.

1981-01-01

Microorganisms and biomolecules exposed to space vacuum and to different intensities of selected wavelengths of solar ultraviolet radiation is studied. The influence of these factors, applied singly or simultaneously, on the integrity of microbial systems and biomolecules is measured. Specifically, this experiment will study in Bacillus subtilis spores (1) disturbances in subsequent germination, outgrowth, and colony formation; (2) photochemical reactions of the DNA and protein in vivo and in vitro and their role in biological injury; and (3) the efficiency of repair processes in these events.

Tumorigenic action of beta, proton, alpha and electron radiation on the rat skin

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

Burns, F.J.

1980-01-01

Rat skin is utilized as a model system for studying dose and time related aspects of the oncogenic action of ionizing radiation, ultraviolet light and polycyclic aromatic hydrocarbons. Molecular lesions in the DNA of the epidermis, including strand breaks and thymine dimers, are measured and compared to the temporal and dose related aspects of tumor induction. The induction and repair kinetics of molecular lesions are compared to split dose recovery as modified by sensitizers and type of radition of oncogenic damage.

Protective effects of a new phloretin derivative against UVB-induced damage in skin cell model and human volunteers.

PubMed

Shin, Seoungwoo; Kum, Hyunwoo; Ryu, Dehun; Kim, Minkyung; Jung, Eunsun; Park, Deokhoon

2014-10-20

The phenolic compound phloretin is a prominent member of the chemical class of dihydrochalcones. Phloretin is specifically found in apple and apple juice and known for its biological properties. We were particularly interested in its potential dermo-cosmetic applications. However, practical limitations of phloretin do exist due to its poor water-solubility. Phloretin was sulfonated with sulfuric acid (98%, wt) and mixed with saturated salt water to produce phloretin 3',3-disulfonate in order to increase its water-solubility. Here we reported the photoprotective effect of phloretin 3',3-disulfonate (PS), a new semi-synthetic derivative of phloretin. Results showed that PS attenuated cyclobutane pyrimidine dimer (CPDs) formation, glutathione (GSH) depletion and apoptosis induced by ultraviolet B (UVB). The photoprotective effect of PS is tightly correlated to the enhancement of nucleotide excision repair (NER) gene expression. Furthemore, PS had inhibitory effects on UVB-induced release of the inflammatory mediators, such as IL-6 and prostaglandin-E2. We also confirmed the safety and clinical efficacy of PS on human skin. Overall, the results demonstrated significant benefits of PS on the protection of keratinocytes against UVB-induced injuries and suggested its potential use in skin photoprotection.

Protective Effects of a New Phloretin Derivative against UVB-Induced Damage in Skin Cell Model and Human Volunteers

PubMed Central

Shin, Seoungwoo; Kum, Hyunwoo; Ryu, Dehun; Kim, Minkyung; Jung, Eunsun; Park, Deokhoon

2014-01-01

The phenolic compound phloretin is a prominent member of the chemical class of dihydrochalcones. Phloretin is specifically found in apple and apple juice and known for its biological properties. We were particularly interested in its potential dermo-cosmetic applications. However, practical limitations of phloretin do exist due to its poor water-solubility. Phloretin was sulfonated with sulfuric acid (98%, wt) and mixed with saturated salt water to produce phloretin 3',3-disulfonate in order to increase its water-solubility. Here we reported the photoprotective effect of phloretin 3',3-disulfonate (PS), a new semi-synthetic derivative of phloretin. Results showed that PS attenuated cyclobutane pyrimidine dimer (CPDs) formation, glutathione (GSH) depletion and apoptosis induced by ultraviolet B (UVB). The photoprotective effect of PS is tightly correlated to the enhancement of nucleotide excision repair (NER) gene expression. Furthemore, PS had inhibitory effects on UVB-induced release of the inflammatory mediators, such as IL-6 and prostaglandin-E2. We also confirmed the safety and clinical efficacy of PS on human skin. Overall, the results demonstrated significant benefits of PS on the protection of keratinocytes against UVB-induced injuries and suggested its potential use in skin photoprotection. PMID:25334063

Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis

PubMed Central

Arora, Sumit; Tyagi, Nikhil; Bhardwaj, Arun; Rusu, Lilia; Palanki, Rohan; Vig, Komal; Singh, Shree R.; Singh, Ajay P.; Palanki, Srinivas; Miller, Michael E.; Carter, James E.; Singh, Seema

2015-01-01

Ultraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis. PMID:25804413

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

PubMed

Alvarado-Cruz, Isabel; Sánchez-Guerra, Marco; Hernández-Cadena, Leticia; De Vizcaya-Ruiz, Andrea; Mugica, Violeta; Pelallo-Martínez, Nadia Azenet; Solís-Heredia, María de Jesús; Byun, Hyang-Min; Baccarelli, Andrea; Quintanilla-Vega, Betzabet

2017-01-01

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene.

PubMed

Drost, Mark; Koppejan, Hester; de Wind, Niels

2013-11-01

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS. © 2013 WILEY PERIODICALS, INC.

Therapeutic Gene Editing Safety and Specificity.

PubMed

Lux, Christopher T; Scharenberg, Andrew M

2017-10-01

Therapeutic gene editing is significant for medical advancement. Safety is intricately linked to the specificity of the editing tools used to cut at precise genomic targets. Improvements can be achieved by thoughtful design of nucleases and repair templates, analysis of off-target editing, and careful utilization of viral vectors. Advancements in DNA repair mechanisms and development of new generations of tools improve targeting of specific sequences while minimizing risks. It is important to plot a safe course for future clinical trials. This article reviews safety and specificity for therapeutic gene editing to spur dialogue and advancement. Copyright © 2017 Elsevier Inc. All rights reserved.

Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3.

PubMed

Lehner, Kevin R; Stone, Megan M; Farber, Rosann A; Petes, Thomas D

2007-11-01

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.

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

NASA Technical Reports Server (NTRS)

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

2003-01-01

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

Prevalence and Penetrance of Major Genes and Polygenes for Colorectal Cancer

PubMed Central

Win, Aung Ko; Jenkins, Mark A.; Dowty, James G.; Antoniou, Antonis C.; Lee, Andrew; Giles, Graham G.; Buchanan, Daniel D.; Clendenning, Mark; Rosty, Christophe; Ahnen, Dennis J.; Thibodeau, Stephen N.; Casey, Graham; Gallinger, Steven; Le Marchand, Loïc; Haile, Robert W.; Potter, John D.; Zheng, Yingye; Lindor, Noralane M.; Newcomb, Polly A.; Hopper, John L.; MacInnis, Robert J.

2016-01-01

Background While high-risk mutations in identified major susceptibility genes (DNA mismatch repair genes and MUTYH) account for some familial aggregation of colorectal cancer, their population prevalence and the causes of the remaining familial aggregation are not known. Methods We studied the families of 5,744 colorectal cancer cases (probands) recruited from population cancer registries in the USA, Canada and Australia and screened probands for mutations in mismatch repair genes and MUTYH. We conducted modified segregation analyses using the cancer history of first-degree relatives, conditional on the proband’s age at diagnosis. We estimated the prevalence of mutations in the identified genes, the prevalence of and hazard ratio for unidentified major gene mutations, and the variance of the residual polygenic component. Results We estimated that 1 in 279 of the population carry mutations in mismatch repair genes (MLH1= 1 in 1946, MSH2= 1 in 2841, MSH6= 1 in 758, PMS2= 1 in 714), 1 in 45 carry mutations in MUTYH, and 1 in 504 carry mutations associated with an average 31-fold increased risk of colorectal cancer in unidentified major genes. The estimated polygenic variance was reduced by 30–50% after allowing for unidentified major genes and decreased from 3.3 for age <40 years to 0.5 for age ≥70 years (equivalent to sibling relative risks of 5.1 to 1.3, respectively). Conclusion Unidentified major genes might explain one-third to one-half of the missing heritability of colorectal cancer. Impact Our findings could aid gene discovery and development of better colorectal cancer risk prediction models. PMID:27799157

The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder.

PubMed

Walden, Helen; Deans, Andrew J

2014-01-01

Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia, a disorder characterized by sensitivity to DNA interstrand crosslinking agents. The clinical features of cytopenia, developmental defects, and tumor predisposition are similar in each group, suggesting that the gene products participate in a common pathway. The Fanconi anemia DNA repair pathway consists of an anchor complex that recognizes damage caused by interstrand crosslinks, a multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several downstream repair proteins including nucleases and homologous recombination enzymes. We review progress in the use of structural and biochemical approaches to understanding how each FANC protein functions in this pathway.

The Fanconi anemia pathway and ICL repair: implications for cancer therapy

PubMed Central

Wang, Lily C; Gautier, Jean

2011-01-01

Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and –independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates (Moldovan and D’Andrea, 2009). However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition. PMID:20807115

Genes on chromosomes 1 and 4 in the mouse are associated with repair of radiation-induced chromatin damage.

PubMed

Potter, M; Sanford, K K; Parshad, R; Tarone, R E; Price, F M; Mock, B; Huppi, K

1988-04-01

Early-passage skin fibroblasts from different inbred and congenic strains of mice were X-irradiated (1 Gy), and the number of chromatid breaks was determined at 2.0 h after irradiation. The cells from DBA/2N, C3H/HeN, STS/A, C57BL/6N, BALB/cJ, and AKR/N had 25 to 42 chromatid breaks per 100 metaphase cells (efficient repair phenotype). NZB/NJ had greater than 78 and BALB/cAn had 87 to 110 chromatid breaks per 100 cells (inefficient repair phenotype). Differences between BALB/cAn and BALB/c. DBA/2 congenic strains which carry less than 1% of the DBA/2 genome indicate that two genes, one on chromosome 1 linked to bcl-2-Pep-3 and the other on chromosome 4 closely linked to Fv-1, affect the efficiency with which the cells repair radiation-induced chromatin damage.

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

PubMed Central

Kornete, Mara

2018-01-01

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing–mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells. PMID:29445007

Targeted mutagenesis in a human-parasitic nematode

PubMed Central

Gang, Spencer S.; Castelletto, Michelle L.

2017-01-01

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites. PMID:29016680

THE ROLE OF THE RETINOBLASTOMA/E2F1 TUMOR SUPPRESSOR PATHWAY IN THE LESION RECOGNITION STEP OF NUCLEOTIDE EXCISION REPAIR

PubMed Central

Lin, Patrick S.; McPherson, Lisa A.; Chen, Aubrey Y.; Sage, Julien; Ford, James M.

2009-01-01

The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb−/−;p107−/−;p130−/− MEFs repaired both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1−/− MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1−/− cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair. PMID:19376752

Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease.

PubMed

Modena, Brian D; Bleecker, Eugene R; Busse, William W; Erzurum, Serpil C; Gaston, Benjamin M; Jarjour, Nizar N; Meyers, Deborah A; Milosevic, Jadranka; Tedrow, John R; Wu, Wei; Kaminski, Naftali; Wenzel, Sally E

2017-06-01

Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. Identify networks of genes reflective of underlying biological processes that define SA. Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.

Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease

PubMed Central

Modena, Brian D.; Bleecker, Eugene R.; Busse, William W.; Erzurum, Serpil C.; Gaston, Benjamin M.; Jarjour, Nizar N.; Meyers, Deborah A.; Milosevic, Jadranka; Tedrow, John R.; Wu, Wei; Kaminski, Naftali

2017-01-01

Rationale: Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. Objectives: Identify networks of genes reflective of underlying biological processes that define SA. Methods: Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. Measurements and Main Results: Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12–21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. Conclusions: In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes. PMID:27984699

Responses of He-Ne laser on agronomic traits and the crosstalk between UVR8 signaling and phytochrome B signaling pathway in Arabidopsis thaliana subjected to supplementary ultraviolet-B (UV-B) stress.

PubMed

Gao, Limei; Li, Yongfeng; Shen, Zhihua; Han, Rong

2018-05-01

UV-B acclimation effects and UV-B damage repair induced by a 632.8-nm He-Ne laser were investigated in Arabidopsis thaliana plants in response to supplementary UV-B stress. There was an increasing trend in growth parameters in the combination-treated plants with He-Ne laser and UV-B light compared to those stressed with enhanced UV-B light alone during different developmental stages of plants. The photosynthetic efficiency (Pn) and survival rates of seedlings were significantly higher in the combination treatments than UV-B stress alone. The expression of UVR8, phytochrome B (PhyB), and their mediated signal responsive genes such as COP1, HY5, and CHS were also significantly upregulated in plants with the laser irradiation compared with other groups without the laser. Levels of flavonol accumulation in leaves and capsule yield of He-Ne laser-treated plants were increased. The phyB-9 mutants were more sensitive to enhanced UV-B stress and had no obvious improvements in plant phenotypic development and physiological damage caused by enhanced UV-B stress after He-Ne laser irradiation. Our results suggested that UVR8 and its mediated signaling pathway via interaction with COP1 can be induced by He-Ne laser, and these processes were dependent on cytoplasmic PhyB levels in plant cells, which might be one of the most important mechanisms of He-Ne laser on UV-B protection and UV-B damage repair. These current data have also elucidated that the biostimulatory effects of He-Ne laser on Arabidopsis thaliana plants would happen not only during the early growth stage but also during the entire late developmental stage.

Elevated Urinary Levels of 8-Hydroxy-2'-deoxyguanosine in a Japanese Child of Xeroderma Pigmentosum/Cockayne Syndrome Complex with Infantile Onset of Nephrotic Syndrome.

PubMed

Kondo, Daiki; Noguchi, Atsuko; Tamura, Hiroaki; Tsuchida, Satoko; Takahashi, Ikuko; Kubota, Hiroki; Yano, Tamami; Oyama, Chikako; Sawaishi, Yukio; Moriwaki, Shinichi; Takahashi, Tsutomu

2016-07-01

Nucleotide excision repair (NER) is an essential biological pathway protecting against ultraviolet light-induced DNA damage. Deficient NER causes a group of rare genetic disorders including two autosomal recessive diseases, xeroderma pigmentosum (XP) and Cockayne syndrome (CS). In addition to the cutaneous photosensitivity shared in XP and CS, CS is featured by growth failure, neurological deterioration, microcephaly, and deep sunken eyes. XP/CS complex is an extremely rare type of NER disorder with a distinct phenotype that is characterized by the skin and eye pathology of XP and the somatic and neurological abnormalities of CS. Some of CS cases have been reported to be complicated with renal failure, but the genetic background or the etiology of the renal failure has not been reported. We herein report a 1-year-old Japanese boy with XP/CS complex, complicated by nephrotic syndrome. Diagnosis was confirmed by the presence of compound heterozygous mutations, G47R (c.139G>A) and R616G (c.1846C>G), in the excision repair cross-complementation group 2 (ERCC2) gene. The kidney biopsies, performed at the age of 1 year and 2 months, revealed diffuse expansion of the mesangial matrix and segmental glomerulosclerosis under light microscopy, and diffused thin capillary walls with partially lamellated regions under electron microscopy. Notably, high levels of urinary 8-hydroxy-2'-deoxyguanosin, known as an oxidative stress marker, were observed during the clinical course. The patient died at the age of 1 year and 11 months because of renal failure. We suggest the involvement of oxidative stress in the pathogenesis of nephrotic syndrome in NER disorders.

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

PubMed

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

2011-11-10

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

UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

PubMed Central

Gyenis, Ákos; Umlauf, David; Újfaludi, Zsuzsanna; Boros, Imre; Ye, Tao; Tora, Làszlò

2014-01-01

Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation. PMID:25058334

Enhanced replication of herpes simplex virus type 1 in human cells

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

Miller, C.S.; Smith, K.O.

1991-02-01

The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate (MMS), methyl methanethiosulfonate (MMTS), ultraviolet light (UV), or gamma radiation (GR)) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of themore » infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes.« less

Hypoxyprobe™ reveals dynamic spatial and temporal changes in hypoxia in a mouse model of endometrial breakdown and repair.

PubMed

Cousins, Fiona L; Murray, Alison A; Scanlon, Jessica P; Saunders, Philippa T K

2016-01-19

Menstruation is the culmination of a cascade of events, triggered by the withdrawal of progesterone at the end of the menstrual cycle. Initiation of tissue destruction and endometrial shedding causes spiral arteriole constriction in the functional layer of the endometrium. Upregulation of genes involved in angiogenesis and immune cell recruitment, two processes that are essential to successful repair and remodelling of the endometrium, both thought to be induced by reduced oxygen has been reported. Evidence for stabilisation/increased expression of the transcriptional regulator hypoxia inducible factor in the human endometrium at menses has been published. The current literature debates whether hypoxia plays an essential role during menstrual repair, therefore this study aims to delineate a role for hypoxia using a sensitive detection method (the Hypoxyprobe™) in combination with an established mouse model of endometrial breakdown and repair. Using our mouse model of menses, during which documented breakdown and synchronous repair occurs in a 24 h timeframe, in combination with the Hypoxyprobe™ detection system, oxygen tensions within the uterus were measured. Immunostaining revealed striking spatial and temporal fluctuations in hypoxia during breakdown and showed that the epithelium is also exposed to hypoxic conditions during the repair phase. Furthermore, time-dependent changes in tissue hypoxia correlated with the regulation of mRNAs encoding for the angiogenic genes vascular endothelial growth factor and stromal derived factor (Cxcl12). Our findings are consistent with a role for focal hypoxia during endometrial breakdown in regulating gene expression during menses. These data have implications for treatment of endometrial pathologies such as heavy menstrual bleeding.

DNA repair and recombination in higher plants: insights from comparative genomics of Arabidopsis and rice.

PubMed

Singh, Sanjay K; Roy, Sujit; Choudhury, Swarup Roy; Sengupta, Dibyendu N

2010-07-21

The DNA repair and recombination (DRR) proteins protect organisms against genetic damage, caused by environmental agents and other genotoxic agents, by removal of DNA lesions or helping to abide them. We identified genes potentially involved in DRR mechanisms in Arabidopsis and rice using similarity searches and conserved domain analysis against proteins known to be involved in DRR in human, yeast and E. coli. As expected, many of DRR genes are very similar to those found in other eukaryotes. Beside these eukaryotes specific genes, several prokaryotes specific genes were also found to be well conserved in plants. In Arabidopsis, several functionally important DRR gene duplications are present, which do not occur in rice. Among DRR proteins, we found that proteins belonging to the nucleotide excision repair pathway were relatively more conserved than proteins needed for the other DRR pathways. Sub-cellular localization studies of DRR gene suggests that these proteins are mostly reside in nucleus while gene drain in between nucleus and cell organelles were also found in some cases. The similarities and dissimilarities in between plants and other organisms' DRR pathways are discussed. The observed differences broaden our knowledge about DRR in the plants world, and raises the potential question of whether differentiated functions have evolved in some cases. These results, altogether, provide a useful framework for further experimental studies in these organisms.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

PubMed

Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

2017-07-01

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

PubMed

Wang, Bangmei; Li, Kunyu; Wang, Amy; Reiser, Michelle; Saunders, Thom; Lockey, Richard F; Wang, Jia-Wang

2015-10-01

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Fanconi anemia (cross)linked to DNA repair.

PubMed

Niedernhofer, Laura J; Lalai, Astrid S; Hoeijmakers, Jan H J

2005-12-29

Fanconi anemia is characterized by hypersensitivity to DNA interstrand crosslinks (ICLs) and susceptibility to tumor formation. Despite the identification of numerous Fanconi anemia (FANC) genes, the mechanism by which proteins encoded by these genes protect a cell from DNA interstrand crosslinks remains unclear. The recent discovery of two DNA helicases that, when defective, cause Fanconi anemia tips the balance in favor of the direct involvement of the FANC proteins in DNA repair and the bypass of DNA lesions.

Genetic variation in DNA repair gene XRCC7 (G6721T) and susceptibility to breast cancer.

PubMed

Nasiri, Meysam; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

2012-08-15

The human XRCC7 is a DNA double-strand break (DSBs) repair gene, involved in non-homologous end joining (NHEJ). It is speculated that DNA DSBs repair have an important role during development of breast cancer. The human XRCC7 is a NHEJ DSBs repair gene. Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. This polymorphism may regulate splicing and cause mRNA instability. In the present study, we specifically investigated whether common G6721T genetic variant of XRCC7 was associated with an altered risk of breast cancer. The present study included 362 females with breast cancer. Age frequency-matched controls (362 persons) were randomly selected from the healthy female blood donors, according to the age distribution of the cases. Using RFLP-PCR based method, the polymorphism of XRCC7 was determined. The TG (OR=1.20, 95% CI: 0.83-1.74, P=0.320) and TT (OR=1.01, 95% CI: 0.67-1.53, P=0.933) genotypes had no significant effect on risk of breast cancer, in comparison with the GG genotype. Our present findings indicate that the TT and TG genotypes were not associated with an altered breast cancer risk. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed

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

2012-04-01

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

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

PubMed Central

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

2012-01-01

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

The Genome of Melanoplus sanguinipes Entomopoxvirus

PubMed Central

Afonso, C. L.; Tulman, E. R.; Lu, Z.; Oma, E.; Kutish, G. F.; Rock, D. L.

1999-01-01

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase β, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae. PMID:9847359

Genomic and Molecular Landscape of DNA Damage Repair Deficiency across The Cancer Genome Atlas.

PubMed

Knijnenburg, Theo A; Wang, Linghua; Zimmermann, Michael T; Chambwe, Nyasha; Gao, Galen F; Cherniack, Andrew D; Fan, Huihui; Shen, Hui; Way, Gregory P; Greene, Casey S; Liu, Yuexin; Akbani, Rehan; Feng, Bin; Donehower, Lawrence A; Miller, Chase; Shen, Yang; Karimi, Mostafa; Chen, Haoran; Kim, Pora; Jia, Peilin; Shinbrot, Eve; Zhang, Shaojun; Liu, Jianfang; Hu, Hai; Bailey, Matthew H; Yau, Christina; Wolf, Denise; Zhao, Zhongming; Weinstein, John N; Li, Lei; Ding, Li; Mills, Gordon B; Laird, Peter W; Wheeler, David A; Shmulevich, Ilya; Monnat, Raymond J; Xiao, Yonghong; Wang, Chen

2018-04-03

DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

PubMed

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

2016-12-15

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Construction of a series of congenic mice with recombinant chromosome 1 regions surrounding the genetic loci for resistance to intracellular parasites (Ity, Lsh, and Bcg), DNA repair responses (Rep-1), and the cytoskeletal protein villin (Vil).

PubMed

Mock, B A; Holiday, D L; Cerretti, D P; Darnell, S C; O'Brien, A D; Potter, M

1994-01-01

The interval of mouse chromosome 1 extending from Idh-1 to Pep-3 harbors the natural resistance gene Ity/Lsh/Bcg; it controls the outcome of infection with Salmonella typhimurium, Leishmania donovani, and several Mycobacterium species. This region also contains a DNA repair gene, Rep-1, which determines the rapidity with which double-strand breaks in chromatin are repaired. BALB/cAnPt and DBA/2N mice differ in their phenotypic expression of these genes. To generate appropriate strains of mice for the study of these genes, a series of 10 C.D2 congenic strains recombinant across a 28-centimorgan interval of mouse chromosome 1 extending from Idh-1 to Pep-3 were derived from crosses of the C.D2-Idh-1 Pep-3 congenic strain back to BALB/cAn. Analyses of these recombinant strains will allow the correlation of biological-immunological phenotypes with defined genetic regions.

Construction of a series of congenic mice with recombinant chromosome 1 regions surrounding the genetic loci for resistance to intracellular parasites (Ity, Lsh, and Bcg), DNA repair responses (Rep-1), and the cytoskeletal protein villin (Vil).

PubMed Central

Mock, B A; Holiday, D L; Cerretti, D P; Darnell, S C; O'Brien, A D; Potter, M

1994-01-01

The interval of mouse chromosome 1 extending from Idh-1 to Pep-3 harbors the natural resistance gene Ity/Lsh/Bcg; it controls the outcome of infection with Salmonella typhimurium, Leishmania donovani, and several Mycobacterium species. This region also contains a DNA repair gene, Rep-1, which determines the rapidity with which double-strand breaks in chromatin are repaired. BALB/cAnPt and DBA/2N mice differ in their phenotypic expression of these genes. To generate appropriate strains of mice for the study of these genes, a series of 10 C.D2 congenic strains recombinant across a 28-centimorgan interval of mouse chromosome 1 extending from Idh-1 to Pep-3 were derived from crosses of the C.D2-Idh-1 Pep-3 congenic strain back to BALB/cAn. Analyses of these recombinant strains will allow the correlation of biological-immunological phenotypes with defined genetic regions. PMID:8262646

Significant accumulation of persistent organic pollutants and dysregulation in multiple DNA damage repair pathways in the electronic-waste-exposed populations.

PubMed

He, Xiaobo; Jing, Yaqing; Wang, Jianhai; Li, Keqiu; Yang, Qiaoyun; Zhao, Yuxia; Li, Ran; Ge, Jie; Qiu, Xinghua; Li, Guang

2015-02-01

Electronic waste (e-waste) has created a worldwide environmental and health problem, by generating a diverse group of hazardous compounds such as persistent organic pollutants (POPs). Our previous studies demonstrated that populations from e-waste exposed region have a significantly higher level of chromosomal aberrancy and incidence of DNA damage. In this study, we further demonstrated that various POPs persisted at a significantly higher concentration in the exposed group than those in the unexposed group. The level of reactive oxygen species and micronucleus rate were also significantly elevated in the exposed group. RNA sequencing analysis revealed 31 genes in DNA damage responses and repair pathways that were differentially expressed between the two groups (Log2 ratio >1 or <-1). Our data demonstrated that both females and males of the exposed group have activated a series of DNA damage response genes; however many important DNA repair pathways have been dysregulated. Expressions of NEIL1/3 and RPA3, which are critical in initiating base pair and nucleotide excision repairs respectively, have been downregulated in both females and males of the exposed group. In contrast, expression of RNF8, an E3 ligase involved in an error prone non-homologous end joining repair for DNA double strand break, was upregulated in both genders of the exposed group. The other genes appeared to be differentially expressed only when the males or females of the two groups were compared respectively. Importantly, the expression of cell cycle regulatory gene CDC25A that has been implicated in multiple kinds of malignant transformation was significantly upregulated among the exposed males while downregulated among the exposed females. In conclusion, our studies have demonstrated significant correlations between e-waste disposing and POPs accumulation, DNA lesions and dysregulation of multiple DNA damage repair mechanisms in the residents of the e-waste exposed region. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases.

PubMed

Bettencourt, Conceição; Hensman-Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas-Gómez, Petra; García-Velázquez, Lizbeth Esmeralda; Alonso-Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J; Jones, Lesley

2016-06-01

The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome-wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single-nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10(-5) ). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10(-5) ) and all SCAs (p = 2.22 × 10(-4) ) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10(-5) ), all in the same direction as in the HD GWAS. We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983-990. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Exploring DNA-binding Proteins with In Vivo Chemical Cross-linking and Mass Spectrometry

PubMed Central

Qiu, Haibo; Wang, Yinsheng

2009-01-01

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins which are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level. PMID:19714816

Chloroplast mutations induced by 9-aminoacridine hydrochloride are independent of the plastome mutator in Oenothera.

PubMed

GuhaMajumdar, M; Baldwin, S; Sears, B B

2004-02-01

Oenothera plants homozygous for the recessive plastome mutator allele ( pm) show chloroplast DNA (cpDNA) mutation frequencies that are about 1,000-fold higher than spontaneous levels. The pm-encoded gene product has been hypothesized to have a function in cpDNA replication, repair and/or mutation avoidance. Previous chemical mutagenesis experiments with the alkylating agent nitroso-methyl urea (NMU) showed a synergistic effect of NMU on the induction of mutations in the pm line, suggesting an interaction between the pm-encoded gene product and one of the repair systems that corrects alkylation damage. The goal of the experiments described here was to examine whether the pm activity extends to the repair of damage caused by non-alkylating mutagens. To this end, the intercalating mutagen, 9-aminoacridine hydrochloride (9AA) was tested for synergism with the plastome mutator. A statistical analysis of the data reported here indicates that the pm-encoded gene product is not involved in the repair of the 9AA-induced mutations. However, the recovery of chlorotic sectors in plants derived from the mutagenized seeds shows that 9AA can act as a mutagen of the chloroplast genome.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

PubMed

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

2017-04-11

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

PubMed Central

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

2017-01-01

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles. PMID:28398335

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

PubMed Central

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

2015-01-01

The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95 % CI 0.63–5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

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

PubMed

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

2015-12-01

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

Prognostic value of DNA repair based stratification of hepatocellular carcinoma

PubMed Central

Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

2016-01-01

Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

Selenium Potentiates Chemotherapeutic Selectivity: Improving Efficacy and Reducing Toxicity

DTIC Science & Technology

2008-04-01

C., et al., Genetic correction of DNA repair-deficient/cancer- prone xeroderma pigmentosum group C keratinocytes. Hum Gene Ther, 2003. 14(10): p...983-96. 4. Muotri, A.R., et al., Complementation of the DNA repair deficiency in human xeroderma pigmentosum group a and C cells by recombinant...survival Keywords: DNA-repair, carboplatin, cisplatin, myelosuppression Abbreviations: Xpc, protein encoded by the xeroderma pigmentosum XPC

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

PubMed

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

2016-02-17

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

PubMed

Kulms, D; Pöppelmann, B; Schwarz, T

2000-05-19

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

PubMed

Rey-Rico, Ana; Cucchiarini, Magali

2016-04-01

Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

PubMed

Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T

2000-02-14

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells.

PubMed

Vélez-Cruz, Renier; Zadorin, Anton S; Coin, Frédéric; Egly, Jean-Marc

2013-01-15

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.

Xeroderma pigmentosum complementation group G associated with Cockayne syndrome

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

Vermeulen, W.; Jaspers, N.G.J.; Bootsma, D.

1993-07-01

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two rare inherited disorders with a clinical and cellular hypersensitivity to the UV component of the sunlight spectrum. Although the two traits are generally considered as clinically and genetically distinct entities, on the biochemical level a defect in the nucleotide excision-repair (NER) pathway is involved in both. Classical CS patients are primarily deficient in the preferential repair of DNA damage in actively transcribed genes, whereas in most XP patients the genetic defect affects both [open quotes]preferential[close quotes] and [open quotes]overall[close quotes] NER modalities. Here the authors report a genetic study of twomore » unrelated, severely affected patients with the clinical characteristics of CS but with a biochemical defect typical of XP. By complementation analysis, using somatic cell fusion and nuclear microinjection of cloned repair genes, they assign these two patients to XP complementation group G, which previously was not associated with CS. This observation extends the earlier identification of two patients with a rare combined XP/CS phenotype within XP complementation groups B and D, respectively. It indicates that some mutations in at least three of the seven genes known to be involved in XP also can result in a picture of partial or even full-blown CS. It is concluded that the syndromes XP and CS are biochemically closely related and may be part of a broader clinical disease spectrum. The authors suggest, as a possible molecular mechanism underlying this relation, that the XPGC repair gene has an additional vital function, as shown for some other NER genes. 33 refs., 5 tabs.« less

Inhibition of microsomal prostaglandin E synthase-1 facilitates liver repair after hepatic injury in mice.

PubMed

Nishizawa, Nobuyuki; Ito, Yoshiya; Eshima, Koji; Ohkubo, Hirotoki; Kojo, Ken; Inoue, Tomoyoshi; Raouf, Joan; Jakobsson, Per-Johan; Uematsu, Satoshi; Akira, Shizuo; Narumiya, Shuh; Watanabe, Masahiko; Majima, Masataka

2018-07-01

Liver repair following hepatic ischemia/reperfusion (I/R) injury is crucial to survival. This study aims to examine the role of endogenous prostaglandin E 2 (PGE 2 ) produced by inducible microsomal PGE synthase-1 (mPGES-1), a terminal enzyme of PGE 2 generation, in liver injury and repair following hepatic I/R. mPGES-1 deficient (Ptges -/- ) mice or their wild-type (WT) counterparts were subjected to partial hepatic ischemia followed by reperfusion. The role of E prostanoid receptor 4 (EP4) was then studied using a genetic knockout model and a selective antagonist. Compared with WT mice, Ptges -/- mice exhibited reductions in alanine aminotransferase (ALT), necrotic area, neutrophil infiltration, chemokines, and proinflammatory cytokine levels. Ptges -/- mice also showed promoted liver repair and increased Ly6C low macrophages (Ly6C low /CD11b high /F4/80 high -cells) with expression of anti-inflammatory and reparative genes, while WT mice exhibited delayed liver repair and increased Ly6C high macrophages (Ly6C high /CD11b high /F4/80 low -cells) with expression of proinflammatory genes. Bone marrow (BM)-derived mPGES-1-deficient macrophages facilitated liver repair with increases in Ly6C low macrophages. In vitro, mPGES-1 was expressed in macrophages polarized toward the proinflammatory profile. Mice treated with the mPGES-1 inhibitor Compound III displayed increased liver protection and repair. Hepatic I/R enhanced the hepatic expression of PGE receptor subtype, EP4, in WT mice, which was reduced in Ptges -/- mice. A selective EP4 antagonist and genetic deletion of Ptger4, which codes for EP4, accelerated liver repair. The proinflammatory gene expression was upregulated by stimulation of EP4 agonist in WT macrophages but not in EP4-deficient macrophages. These results indicate that mPGES-1 regulates macrophage polarization as well as liver protection and repair through EP4 signaling during hepatic I/R. Inhibition of mPGES-1 could have therapeutic potential by promoting liver repair after acute liver injury. Hepatic ischemia/reperfusion injury is a serious complication that occurs in liver surgery. Herein, we demonstrated that inducible prostaglandin E 2 synthase (mPGES-1), an enzyme involved in synthesizing prostaglandin E 2 , worsens the injury and delays liver repair through accumulation of proinflammatory macrophages. Inhibition of mPGES-1 offers a potential therapy for both liver protection and repair in hepatic ischemia/reperfusion injury. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Multiple cutaneous malignancies in a patient of xeroderma pigmentosum.

PubMed

Grampurohit, Vandana U; Dinesh, U S; Rao, Ravikala

2011-01-01

Xeroderma pigmentosum is a genodermatosis characterized by photosensitivity and the development of cutaneous and internal malignancies at an early age. The basic defect underlying the clinical manifestations is a nucleotide excision repair defect, leading to defective repair of DNA damaged by ultraviolet radiation. These patients exhibit enhanced sensitivity to ionizing radiation. Patients with xeroderma pigmentosum who are younger than 20 years of age have a greater than 1000-fold increased risk of developing skin cancer. Early detection of these malignancies is necessary because they are fast growing, metastasize early and lead to death. Although, early detection and treatment of cutaneous malignancies will reduce the morbidity and mortality, genetic counseling remains the most important measure for preventing xeroderma pigmentosum. We report a case of xeroderma pigmentosum in an 18-year-old male presenting with multiple cutaneous malignancies: squamous cell carcinoma, malignant melanoma and pigmented basal cell carcinoma.

Xeroderma Pigmentosum - Facts and Perspectives.

PubMed

Lehmann, Janin; Seebode, Christina; Martens, Marie Christine; Emmert, Steffen

2018-02-01

Ultraviolet (UV)-induced DNA lesions are almost exclusively removed by the nucleotide excision repair (NER) pathway, which is essential for prevention of skin cancer development. Patients with xeroderma pigmentosum (XP) are extremely sun sensitive due to a genetic defect in components of the NER cascade. They present with first signs of premature skin aging at an early age, with a considerably increased risk of developing UV-induced skin cancer. XP belongs to the group of DNA repair defective disorders that are mainly diagnosed in the clinic and in hindsight confirmed at the molecular level. Unfortunately, there are no causative treatment options for this rare, autosomal-recessive disorder, emphasizing the importance of an early diagnosis. Subsequently, UV-protective measures such as the reduction of exposure to environmental UV and regular skin cancer screenings should be undertaken to substantially improve prognosis as well as the disease course. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

How Magnetotactic Bacteria Respond to Radiation Induced Stress and Damage: Comparative Genomics Evidences for Evolutionary Adaptation

NASA Astrophysics Data System (ADS)

Wang, Y.; Pan, Y.

2015-12-01

Solar radiation and galactic cosmic radiation is believed to be major restriction factors influencing survival and evolution of life. On planet earth, geomagnetic field along with atmosphere protect living beings from the harmful radiation. During a geomagnetic reversal or excursion, however, the efflux of charged particles on earth surface would increase as the shielding effect of magnetic field decrease. The stratospheric ozone can also be partially stripped away by solar wind when the strength of the field is weak, leading to an increasing ultraviolet radiation penetration to the earth surface. However, studies on the mechanism of radiation induced stress and damage are focused only on bacteria that have no response to magnetic field. This study was motivated by the need to fill the gap upon knowledge of that on magnetic field sensitive microorganism. Magnetotactic bacteria (MTB) are a group of microbes that are able to synthesis intracellular nano-sized magnetic particles (named magnetosomes). These chain-arranged magnetosomes help MTB sense and swim along the magnetic field to find their optimal living environment efficiently. In this paper, in silico prediction of stress and damage repair genes in response to different radiation were carried out on the complete genome of four nonmagnetotactic and four magnetotactic spirilla. In silico analyses of the genomes of magnetic field sensitive and non-sensitive spirilla revealed: 1) all strains contain genes for regulate responses superoxide and peroxide stress, DNA pyrimidine dimer and string breaks; 2) non-magnetotactic spirilla have more genes dealing with oxidative stress, while magnetotactic spirilla may benefit from magnetotaxis by swimming into oxic-anoxic zone away from oxidative stress and direct radiation damage; yet, the lipid hydroperoxide peroxidase gene in MTB may be responsible for possible ROS generated by the membrane enveloped magnetite magnetosome; 3) magnetotactic spirilla possess SOS recA mediated umuCD genes and double copied ssb gene, these low fidelity DNA polymerase along with Ssb protein may endow MTB high adaptive mutation under stress condition; 4) also, magnetosome crystals (magnetite or greigite) can reduce radiation oxidative damage and protect MTB.

Methylation profile analysis of DNA repair genes in hepatocellular carcinoma with MS-MLPA.

PubMed

Ozer, Ozge; Bilezikci, Banu; Aktas, Sema; Sahin, Feride I

2013-12-01

Hepatocellular carcinoma (HCC) is one of the rare tumors with well-defined risk factors. The multifactorial etiology of HCC can be explained by its complex molecular pathogenesis. In the current study, the methylation status of 7 genes involved in DNA repair mechanisms, namely MLH1, PMS2, MSH6, MSH2, MGMT, MSH3, and MLH3, was investigated in tumor samples from HCC patients, using the methylation-specific-multiplex ligated probe amplification method and the results were correlated with available clinical findings. The most common etiological factor in these cases was the presence of hepatitis B alone (47.2%). Among the 56 cases that were studied, promoter methylation was detected in at least one of the genes in 27 (48.2%) cases, only in 1 gene in 13 (23.2%) cases, and in >1 gene in 14 (25%) cases. Of the 7 genes investigated, methylation was most frequently observed in MSH3, in 14 (25%) cases. Methylation of at least 1 gene was significantly more frequent in patients with single tumors than multifocal tumors. There were significant differences regarding hepatitis B status, Child Class, tumor number, grade, and TNM stage in cases where PMS2 methylation was detected. Our results suggest that methylation of genes involved in mismatch repair may be responsible in the pathogenesis of HCC, and evaluating changes in multiple genes in these pathways simultaneously would be more informative. Despite being a robust and relatively inexpensive method, the methylation-specific-multiplex ligated probe amplification assay could be more extensively applied with improvements in the currently intricate data analysis component.

The Fanconi anemia protein interaction network: casting a wide net.

PubMed

Rego, Meghan A; Kolling, Frederick W; Howlett, Niall G

2009-07-31

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J. Fornace, Jr., J.B. Little, R.R. Weichselbaum, DNA repair in a Fanconi's anemia fibroblast cell strain, Biochim. Biophys. Acta 561 (1979) 99-109; Y. Fujiwara, M. Tatsumi, Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun. 66 (1975) 592-598; A.J. Rainbow, M. Howes, Defective repair of ultraviolet- and gamma-ray-damaged DNA in Fanconi's anaemia, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 31 (1977) 191-195]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [A.D. Auerbach, Fanconi anemia diagnosis and the diepoxybutane (DEB) test, Exp. Hematol. 21 (1993) 731-733]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase.

PubMed

Tanaka, Miyuki; Takahara, Michiyo; Nukina, Kohei; Hayashi, Akiyo; Sakai, Wataru; Sugasawa, Kaoru; Shiomi, Yasushi; Nishitani, Hideo

2017-04-03

Cdt1 is rapidly degraded by CRL4 Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2 . The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4 Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, February 1, 1993--November 1, 1994

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

Hawley, R.S.

This research focuses on two repair deficient mutations in Drosophila melanogaster, namely mei-9, mei-41. In addition, the authors propose to extend this study to include the mus-312 mutation. They expect these studies to provide substantial insights into both the molecular mechanisms of DNA repair in Drosophila and the role these genes play in normal biological processes.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

NASA Astrophysics Data System (ADS)

Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p53R2 gene expression modulations shows a response lasting up to 24 hours after irradiation.

Photomorphogenic responses to ultraviolet-B light.

PubMed

Jenkins, Gareth I

2017-11-01

Exposure to ultraviolet B (UV-B) light regulates numerous aspects of plant metabolism, morphology and physiology through the differential expression of hundreds of genes. Photomorphogenic responses to UV-B are mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8). Considerable progress has been made in understanding UVR8 action: the structural basis of photoreceptor function, how interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 initiates signaling and how REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins negatively regulate UVR8 action. In addition, recent research shows that UVR8 mediates several responses through interaction with other signaling pathways, in particular auxin signaling. Nevertheless, many aspects of UVR8 action remain poorly understood. Most research to date has been undertaken with Arabidopsis, and it is important to explore the functions and regulation of UVR8 in diverse plant species. Furthermore, it is essential to understand how UVR8, and UV-B signaling in general, regulates processes under natural growth conditions. Ultraviolet B regulates the expression of many genes through UVR8-independent pathways, but the activity and importance of these pathways in plants growing in sunlight are poorly understood. © 2017 John Wiley & Sons Ltd.

Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis.

PubMed

Davila, Jaime I; Arrieta-Montiel, Maria P; Wamboldt, Yashitola; Cao, Jun; Hagmann, Joerg; Shedge, Vikas; Xu, Ying-Zhi; Weigel, Detlef; Mackenzie, Sally A

2011-09-27

The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. We obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

Single nucleotide polymorphisms of DNA repair genes as predictors of radioresponse.

PubMed

Parliament, Matthew B; Murray, David

2010-10-01

Radiation therapy is a key modality in the treatment of cancer. Substantial progress has been made in unraveling the molecular events which underpin the responses of malignant and surrounding normal tissues to ionizing radiation. An understanding of the genes involved in processes such as DNA double-strand break repair, DNA damage response, cell-cycle control, apoptosis, cellular antioxidant defenses, and cytokine production, has evolved toward examination of how genetic variants, most often, single nucleotide polymorphisms (SNPs), may influence interindividual radioresponse. Experimental approaches, such as candidate SNP-association studies, genome-wide association studies, and massively parallel sequencing are being proposed to address these questions. We present a focused review of the evidence supporting an association between SNPs in DNA repair genes and radioresponse in normal tissues and tumors. Although preliminary results indicate possible associations, there are methodological weaknesses in many of the studies, and independent validation of SNPs as biomarkers of radioresponse in much larger cohorts will likely require research cooperation through international consortia. Copyright © 2010 Elsevier Inc. All rights reserved.

Genetic polymorphisms of genes involved in DNA repair and metabolism influence micronucleus frequencies in human peripheral blood lymphocytes.

PubMed

Dhillon, Varinderpal S; Thomas, Philip; Iarmarcovai, G; Kirsch-Volders, Micheline; Bonassi, Stefano; Fenech, Michael

2011-01-01

The cytokinesis-block micronucleus cytome (CBMNCyt) assay is a widely used technique for measuring DNA damage in human populations. The formation of micronuclei (MN) in dividing cells can result from chromosome breakage due to unrepaired or mis-repaired DNA lesions or chromosome malsegregation due to mitotic malfunction. The sensitivity of the MN assay to polymorphisms in various genes involved in DNA repair, activation/deactivation of carcinogens/chemicals/drugs/alcohol, folate metabolism pathway and micronutrient transport has been extensively reported in the literature. MN frequency is also an important index for determining DNA repair efficiency phenotype (including mis-repair), response to environmental exposure and identifying various dietary factors required for optimal genome stability. The aim of the present study is to review the reported in vivo associations between genotype and MN frequency in humans taking into considerations the presence of interactions with nutrients levels and/or exposure to genotoxins. One hundred and eleven publications linking MN frequency in peripheral blood lymphocytes to gene polymorphism were retrieved from PubMed. After applying exclusion criteria, only 37 studies were evaluated in the present review. Polymorphisms in XRCC1 (Arg280His), ERCC2 (Lys751Gln), CYP2E1 (c1/c2) and MTR (A2756G) were consistently associated with the MN formation. These results contribute substantial evidence to the hypothesis that genotype may influence MN frequency in human cells.

Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH).

PubMed Central

van Vuuren, A J; Vermeulen, W; Ma, L; Weeda, G; Appeldoorn, E; Jaspers, N G; van der Eb, A J; Bootsma, D; Hoeijmakers, J H; Humbert, S

1994-01-01

ERCC3 was initially identified as a gene correcting the nucleotide excision repair (NER) defect of xeroderma pigmentosum complementation group B (XP-B). The recent finding that its gene product is identical to the p89 subunit of basal transcription factor BTF2(TFIIH), opened the possibility that it is not directly involved in NER but that it regulates the transcription of one or more NER genes. Using an in vivo microinjection repair assay and an in vitro NER system based on cell-free extracts we demonstrate that ERCC3 in BTF2 is directly implicated in excision repair. Antibody depletion experiments support the idea that the p62 BTF2 subunit and perhaps the entire transcription factor function in NER. Microinjection experiments suggest that exogenous ERCC3 can exchange with ERCC3 subunits in the complex. Expression of a dominant negative K436-->R ERCC3 mutant, expected to have lost all helicase activity, completely abrogates NER and transcription and concomitantly induces a dramatic chromatin collapse. These findings establish the role of ERCC3 and probably the entire BTF2 complex in transcription in vivo which was hitherto only demonstrated in vitro. The results strongly suggest that transcription itself is a critical component for maintenance of chromatin structure. The remarkable dual role of ERCC3 in NER and transcription provides a clue in understanding the complex clinical features of some inherited repair syndromes. Images PMID:8157004

Micro-RNAs in regenerating lungs: an integrative systems biology analysis of murine influenza pneumonia.

PubMed

Tan, Kai Sen; Choi, Hyungwon; Jiang, Xiaoou; Yin, Lu; Seet, Ju Ee; Patzel, Volker; Engelward, Bevin P; Chow, Vincent T

2014-07-11

Tissue regeneration in the lungs is gaining increasing interest as a potential influenza management strategy. In this study, we explored the role of microRNAs, short non-coding RNAs involved in post-transcriptional regulation, during pulmonary regeneration after influenza infection. We profiled miRNA and mRNA expression levels following lung injury and tissue regeneration using a murine influenza pneumonia model. BALB/c mice were infected with a sub-lethal dose of influenza A/PR/8(H1N1) virus, and their lungs were harvested at 7 and 15 days post-infection to evaluate the expression of ~300 miRNAs along with ~36,000 genes using microarrays. A global network was constructed between differentially expressed miRNAs and their potential target genes with particular focus on the pulmonary repair and regeneration processes to elucidate the regulatory role of miRNAs in the lung repair pathways. The miRNA arrays revealed a global down-regulation of miRNAs. TargetScan analyses also revealed specific miRNAs highly involved in targeting relevant gene functions in repair such as miR-290 and miR-505 at 7 dpi; and let-7, miR-21 and miR-30 at 15 dpi. The significantly differentially regulated miRNAs are implicated in the activation or suppression of cellular proliferation and stem cell maintenance, which are required during the repair of the damaged lungs. These findings provide opportunities in the development of novel repair strategies in influenza-induced pulmonary injury.

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

PubMed

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

2014-06-01

The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Discovery of mutations in homologous recombination genes in African-American women with breast cancer.

PubMed

Ding, Yuan Chun; Adamson, Aaron W; Steele, Linda; Bailis, Adam M; John, Esther M; Tomlinson, Gail; Neuhausen, Susan L

2018-04-01

African-American women are more likely to develop aggressive breast cancer at younger ages and experience poorer cancer prognoses than non-Hispanic Caucasians. Deficiency in repair of DNA by homologous recombination (HR) is associated with cancer development, suggesting that mutations in genes that affect this process may cause breast cancer. Inherited pathogenic mutations have been identified in genes involved in repairing DNA damage, but few studies have focused on African-Americans. We screened for germline mutations in seven HR repair pathway genes in DNA of 181 African-American women with breast cancer, evaluated the potential effects of identified missense variants using in silico prediction software, and functionally characterized a set of missense variants by yeast two-hybrid assays. We identified five likely-damaging variants, including two PALB2 truncating variants (Q151X and W1038X) and three novel missense variants (RAD51C C135R, and XRCC3 L297P and V337E) that abolish protein-protein interactions in yeast two-hybrid assays. Our results add to evidence that HR gene mutations account for a proportion of the genetic risk for developing breast cancer in African-Americans. Identifying additional mutations that diminish HR may provide a tool for better assessing breast cancer risk and improving approaches for targeted treatment.

Antimutagenic, antigenotoxic and antioxidant activities of Acacia salicina extracts (ASE) and modulation of cell gene expression by H2O2 and ASE treatment.

PubMed

Bouhlel, Ines; Valenti, Kita; Kilani, Soumaya; Skandrani, Ines; Ben Sghaier, Mohamed; Mariotte, Anne-Marie; Dijoux-Franca, Marie-Genevieve; Ghedira, Kamel; Hininger-Favier, Isabelle; Laporte, François; Chekir-Ghedira, Leila

2008-08-01

The total oligomers flavonoids (TOF), chloroform, petroleum ether and aqueous extracts from Acacia salicina, were investigated for the antioxidative, cytotoxic, antimutagenic and antigenotoxic activities. The viability of K562 cells were affected by all extracts after 48 h exposure. Our results showed that A. salicina extracts have antigenotoxic and/or antimutagenic activities. TOF and chloroform extracts exhibit antioxidant properties, expressed by the capacity of these extracts to inhibit xanthine oxidase activity. To further explore the mechanism of action of A. salicina extracts, we characterized expression profiles of genes involved in antioxidant protection and DNA repair in the human lymphoblastic cell line K562 exposed to H2O2. Transcription of several genes related to the thioredoxin antioxidant system and to the DNA base-excision repair pathway was up-regulated after incubation with chloroform, TOF and petroleum ether extracts. Moreover genes involved in the nucleotide-excision repair pathway and genes coding for catalase and Mn-superoxide-dismutase, two important antioxidant enzymes, were induced after incubation with the chloroform extract. Taken together, these observations provide evidence that the chloroform and TOF extracts of A. salicina leaves contain bioactive compounds that are able to protect cells against the consequences of an oxidative stress.

Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping

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

Miskin, R.; Reich, E.; Dixon, K.

1981-10-01

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agentsmore » induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.« less

Repair genes expression profile of MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers.

PubMed

Alves, Mônica Ghislaine Oliveira; Carta, Celina Faig Lima; de Barros, Patrícia Pimentel; Issa, Jaqueline Scholz; Nunes, Fábio Daumas; Almeida, Janete Dias

2017-01-01

The aim of this study was to evaluate the effect of chronic smoking on the expression profile of the repair genes MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers and never smokers. The sample consisted of thirty exfoliative cytology smears per group obtained from Smokers and Never Smokers. Total RNA was extracted and expression of the MLH1, MSH2 and ATM genes were evaluated by quantitative real-time and immunocytochemistry. The gene and protein expression data were correlated to the clinical data. Gene expression was analyzed statistically using the Student t-test and Pearson's correlation coefficient, with p<0.05. MLH1, MSH2 and ATM genes were downregulated in the smoking group compared to the control with significant values for MLH1 (p=0.006), MSH2 (p=0.0001) and ATM (p=0.0001). Immunocytochemical staining for anti-MLH1, anti-MSH2 and anti-ATM was negative in Never Smokers; in Smokers it was rarely positive. No significant correlation was observed among the expression of MLH1, MSH2, ATM and age, number of cigarettes consumed per day, time of smoking during life, smoking history or levels of CO in expired air. The expression of genes and proteins related to DNA repair mechanism MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers was reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mismatch repair deficiency associated with overexpression of the MSH3 gene.

PubMed

Marra, G; Iaccarino, I; Lettieri, T; Roscilli, G; Delmastro, P; Jiricny, J

1998-07-21

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

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

PubMed

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

2018-03-06

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

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair

PubMed Central

Reumann, Marie K.; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Steven B.; Lukashova, Lyudmila; Boskey, Adele L.; Mayer-Kuckuk, Philipp

2011-01-01

Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1−/− mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1−/− mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1−/− callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. PMID:21726677

Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

PubMed Central

Haber, James E.

2016-01-01

Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6. PMID:27074148

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

PubMed

Reumann, Marie K; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Stephen B; Lukashova, Lyudmila; Boskey, Adele L; Mayer-Kuckuk, Philipp

2011-10-01

Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Association of Arg194Trp, Arg280His and Arg399Gln Polymorphisms in X-ray Repair Cross-Complementing Group 1 Gene and Risk of Differentiated Thyroid Carcinoma in Iran

PubMed Central

Fard-Esfahani, Pezhman; Fard-Esfahani, Armaghan; Fayaz, Shima; Ghanbarzadeh, Bahareh; Saidi, Parinaz; Mohabati, Reyhaneh; Bidoki, Seyed Kazem; Majdi, Mina

2011-01-01

Background: X-ray repair cross-complementing group 1 (XRCC1) gene is a DNA repair gene and its non-synonymous single nucleotide polymorphisms (SNP) may influence DNA repair capacity which has been considered as a modifying risk factor for cancer development. Methods: A case-control study was conducted to investigate impact of three frequently studied polymorphisms (Arg194Trp, Arg280His and Arg399Gln) on developing differentiated thyroid carcinoma (DTC). Results: Increased risks for DTC were shown in homozygous (odds ratio [OR]: 3.66, 95% confidence interval [CI]: 0.38-35.60) and in dominant trait (OR: 1.22, 95% CI: 1.64-2.32) of Arg194Trp genotype. Also, for Arg280His genotype, an increased risk for DTC was shown in dominant trait (OR: 1.42, 95% confidence interval [CI]: 0.76-2.68), while a mildly reduction of risk for DTC (OR: 0.77, 95% [CI]: 0.50-1.17) was estimated in dominant Gln genotype of Arg399Gln. Considering combinatory effects of Arg194Trp and Arg280His genotypes on DTC, the calculated OR and 95% CI for being heterozygous for one of Arg194Trp or Arg280His genotypes were 1.57 and 0.90-2.74, respectively. Conclusion: Genotyping of codons 194, 280 and 399 in XRCC1 gene may use in risk assessment of DTC. PMID:21987112

Skeletal muscle repair in a mouse model of nemaline myopathy

PubMed Central

Sanoudou, Despina; Corbett, Mark A.; Han, Mei; Ghoddusi, Majid; Nguyen, Mai-Anh T.; Vlahovich, Nicole; Hardeman, Edna C.; Beggs, Alan H.

2012-01-01

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles. PMID:16877500

Skeletal muscle repair in a mouse model of nemaline myopathy.

PubMed

Sanoudou, Despina; Corbett, Mark A; Han, Mei; Ghoddusi, Majid; Nguyen, Mai-Anh T; Vlahovich, Nicole; Hardeman, Edna C; Beggs, Alan H

2006-09-01

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.

Science of photobiology

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

Smith, K.C.

1974-01-01

Formation of the American Society for Photobiology for meeting the needs of investigators studying the effects of light on man and other organisms is described. The scientific program of the first meeting of the society included discussions of the followiug topics: detrimental effects of excessive exposure to sunlight and artificial uv radiation; repair of uv-induced damage to cells; the roles of light in the human environnnent; photochemistry in photobiology; effects of near uv light; photosynthesis; bioluminescence; light perception without eyes; chronobiology; the ultraviolet world of insects; vision; and solar energy conversion. (HLW)

Induction of polypeptides in Saccharomyces cerevisiae after ultraviolet irradiation.

PubMed

Angulo, J F; Schwencke, J; Fernandez, I; Moustacchi, E

1986-07-31

Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis. UV-irradiation induced the synthesis of various proteins. Among them the analogue of the RecA protein of Escherichia coli (Angulo et al. 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM

EPA Science Inventory

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanism
Anuradha Mudipalli.

Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

Genes on chromosomes 1 and 4 in the mouse are associated with repair of radiation-induced chromatin damage

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

Potter, M.; Sanford, K.K.; Parshad, R.

Early-passage skin fibroblasts from different inbred and congenic strains of mice were X-irradiated (1 Gy), and the number of chromatid breaks was determined at 2.0 h after irradiation. The cells from DBA/2N, C3H/HeN, STS/A, C57BL/6N, BALB/cJ, and AKR/N had 25 to 42 chromatid breaks per 100 metaphase cells (efficient repair phenotype). NZB/NJ had greater than 78 and BALB/cAn had 87 to 110 chromatid breaks per 100 cells (inefficient repair phenotype). Differences between BALB/cAn and BALB/c. DBA/2 congenic strains which carry less than 1% of the DBA/2 genome indicate that two genes, one on chromosome 1 linked to bcl-2-Pep-3 and themore » other on chromosome 4 closely linked to Fv-1, affect the efficiency with which the cells repair radiation-induced chromatin damage.« less

UNG protects B cells from AID-induced telomere loss

PubMed Central

Cortizas, Elena M.; Zahn, Astrid; Safavi, Shiva

2016-01-01

Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG). In contrast, in the absence of UNG activity, deleterious processing by mismatch repair leads to telomere loss and defective cell proliferation. Indeed, we show that UNG deficiency reduces B cell clonal expansion in the germinal center in mice and blocks the proliferation of tumor B cells expressing AID. We propose that AID-induced damage at telomeres acts as a fail-safe mechanism to limit the tumor promoting activity of AID when it overwhelms uracil excision repair. PMID:27697833

Solar ultraviolet radiation and its impact on aquatic systems of Patagonia, South America.

PubMed

Villafañe, V E; Helbling, E W; Zagarese, H E

2001-03-01

Solar ultraviolet radiation (UVR, 280-400 nm) is known to cause a number of detrimental effects in aquatic organisms. The area of Patagonia, which is sometimes under the influence of the Antarctic ozone "hole", occasionally receives enhanced levels of ultraviolet B radiation (UV-B, 280-315 nm). Great efforts have been put into creating a database for UVR climatology by installing a variety of instruments in several localities in the region. However, no comparable effort has been made to determine the impact of normal and enhanced levels of solar UVR upon organisms. Most of the photobiological research in aquatic systems of Patagonia has focused on determining the effects of solar UVR in phytoplankton photosynthesis, DNA damage, and mortality, fecundity and repair mechanisms in zooplanktonic species. Some work has also been done with fish larvae and interactions between species at low trophic levels of the aquatic food web. The results of these studies indicate that in order to assess the overall impact of UVR in a certain waterbody, it is also necessary to consider other variables, such as changes in cloudiness, ozone concentrations, differential sensitivity of organisms, and depth of the upper mixed layer/epilimnion. All factors that can preclude or benefit the acclimation of species to solar radiation.

Acrylate-induced allergic contact dermatitis in a car windscreen repairer.

PubMed

Fremlin, G; Sansom, J

2014-10-01

We report a case of an allergic skin reaction to ultraviolet-cured acrylates in a windscreen repair worker. The patient presented with a 6 month history of fingertip dryness, vesicles and desquamation. He had worked as a self-employed car windscreen repairer for 19 years. Previous management with vinyl glove protection and treatment with clobetasol propionate ointment had produced little improvement. He was patch tested to the British Society for Cutaneous Allergy standard and preservatives series and to the two acrylates used in his work environment, identified using safety data sheets, methyl methacrylate 2% pet and 2-hydroxyethylmethacrylate (2-HEMA) 2% pet. A positive reaction was seen at Day 4 to 2-HEMA, but all other patch tests were negative. An occupational allergic contact dermatitis to 2-HEMA was diagnosed. The patient was given avoidance advice and advised to use nitrile gloves. Although he was unable to give up his current work, he has continued his job using nitrile gloves with marked improvement. © The Author 2014. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress

PubMed Central

Lee, Ming-Hsiang; Wang, Liya; Chang, Zee-Fen

2014-01-01

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state. PMID:24561807

The mutation studies of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts

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

Schultz, R.A.; Chang, C.C.; Trosko, J.E.

1981-01-01

We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to themore » parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis.« less

Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

PubMed Central

2011-01-01

Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches. Results L. hongkongensis possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two arc gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na+:H+ antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs. Conclusions The L. hongkongensis genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals. PMID:21711489

Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome.

PubMed

Westdorp, Harm; Kolders, Sigrid; Hoogerbrugge, Nicoline; de Vries, I Jolanda M; Jongmans, Marjolijn C J; Schreibelt, Gerty

2017-09-10

Monoallelic germline mutations in one of the DNA mismatch repair (MMR) genes cause Lynch syndrome, with a high lifetime risks of colorectal and endometrial cancer at adult age. Less well known, is the constitutional mismatch repair deficiency (CMMRD) syndrome caused by biallelic germline mutations in MMR genes. This syndrome is characterized by the development of childhood cancer. Patients with CMMRD are at extremely high risk of developing multiple cancers including hematological, brain and intestinal tumors. Mutations in MMR genes impair DNA repair and therefore most tumors of patients with CMMRD are hypermutated. These mutations lead to changes in the translational reading frame, which consequently result in neoantigen formation. Neoantigens are recognized as foreign by the immune system and can induce specific immune responses. The growing evidence on the clinical efficacy of immunotherapies, such as immune checkpoint inhibitors, offers the prospect for treatment of patients with CMMRD. Combining neoantigen-based vaccination strategies and immune checkpoint inhibitors could be an effective way to conquer CMMRD-related tumors. Neoantigen-based vaccines might also be a preventive treatment option in healthy biallelic MMR mutation carriers. Future studies need to reveal the safety and efficacy of immunotherapies for patients with CMMRD. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Genetic Causes of Microcephaly and Lessons for Neuronal Development

PubMed Central

Gilmore, Edward C.; Walsh, Christopher A.

2012-01-01

The study of human developmental microcephaly is providing important insights into brain development. It has become clear that developmental microcephalies are associated with abnormalities in cellular production, and that the pathophysiology of microcephaly provides remarkable insights into how the brain generates the proper number of neurons that determine brain size. Most of the genetic causes of ‘primary’ developmental microcephaly (i.e., not associated with other syndromic features) are associated with centrosomal abnormalities. In addition to other functions, centrosomal proteins control the mitotic spindle, which is essential for normal cell proliferation during mitosis. However, the brain is often uniquely affected when microcephaly genes are mutated implying special centrosomal related functions in neuronal production. Although models explaining how this could occur have some compelling data, they are not without controversy. Interestingly, some of the microcephaly genes show evidence that they were targets of evolutionary selection in primates and human ancestors, suggesting potential evolutionary roles in controlling neuronal number and brain volume across species. Mutations in DNA repair pathway genes also lead to microcephaly. Double stranded DNA breaks appear to be a prominent type of damage that needs to be repaired during brain development, yet why defects in DNA repair affect the brain preferentially and if DNA repair relates to centrosome function, are not clearly understood. PMID:24014418

Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki

PubMed Central

Harrison, Neale

2016-01-01

Neuron glia antigen 2 (NG2)–positive glia are repair cells that proliferate upon central nervous system (CNS) damage, promoting functional recovery. However, repair is limited because of the failure of the newly produced glial cells to differentiate. It is a key goal to discover how to regulate NG2 to enable glial proliferation and differentiation conducive to repair. Drosophila has an NG2 homologue called kon-tiki (kon), of unknown CNS function. We show that kon promotes repair and identify the underlying mechanism. Crush injury up-regulates kon expression downstream of Notch. Kon in turn induces glial proliferation and initiates glial differentiation by activating glial genes and prospero (pros). Two negative feedback loops with Notch and Pros allow Kon to drive the homeostatic regulation required for repair. By modulating Kon levels in glia, we could prevent or promote CNS repair. Thus, the functional links between Kon, Notch, and Pros are essential for, and can drive, repair. Analogous mechanisms could promote CNS repair in mammals. PMID:27551055

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

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

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

Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase.

PubMed

Murphy, Anar K; Tammaro, Margaret; Cortazar, Frank; Gindt, Yvonne M; Schelvis, Johannes P M

2008-11-27

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.