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Sample records for excision repair enzymes

  1. Response of base excision repair enzymes to complex DNA lesions.

    PubMed

    Weinfeld, M; Rasouli-Nia, A; Chaudhry, M A; Britten, R A

    2001-11-01

    There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions--single-strand breaks or modified nucleosides--located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

  2. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  3. Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes.

    PubMed

    Beard, Brian C; Wilson, Samuel H; Smerdon, Michael J

    2003-06-24

    The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase beta (pol beta) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates ( approximately 10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing "toward" and "away from" the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol beta is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur "unassisted" in chromatin, whereas downstream factors in this pathway (i.e., pol beta) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.

  4. The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

    PubMed Central

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J.

    2012-01-01

    DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894

  5. Base Excision Repair and Cancer

    PubMed Central

    Wallace, Susan S.; Murphy, Drew L.; Sweasy, Joann B.

    2012-01-01

    Base excision repair is the system used from bacteria to man to remove the tens of thousands of endogenous DNA damages produced daily in each human cell. Base excision repair is required for normal mammalian development and defects have been associated with neurological disorders and cancer. In this paper we provide an overview of short patch base excision repair in humans and summarize current knowledge of defects in base excision repair in mouse models and functional studies on short patch base excision repair germ line polymorphisms and their relationship to cancer. The biallelic germ line mutations that result in MUTYH-associated colon cancer are also discussed. PMID:22252118

  6. Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF.

    PubMed

    Tian, Ming; Shinkura, Reiko; Shinkura, Nobuhiko; Alt, Frederick W

    2004-02-01

    Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5' to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.

  7. Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

    PubMed

    Bissenbaev, Amangeldy K; Ishchenko, Alexander A; Taipakova, Sabira M; Saparbaev, Murat K

    2011-10-01

    Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

  8. The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

    SciTech Connect

    Tanihigashi, Haruna; Yamada, Ayako; Igawa, Emi; Ikeda, Shogo . E-mail: ikeda@dbc.ous.ac.jp

    2006-09-08

    In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-{alpha},{beta}-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1{delta} cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1{delta} cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2{delta} cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1{delta} and apn2{delta} cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.

  9. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  10. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    PubMed

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases.

  11. Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate: New Evidence on the Role of PARP-1 in DNA Repair

    SciTech Connect

    Lavrik, Olga I.; Prasad, Rajendra; Sobol, Robert W.; Horton, Julie K.; Ackerman, Eric J. ); Wilson, Samuel H.

    2001-07-06

    To examine mammalian base excision repair (BER) enzymes interacting with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast (MEF) crude extract. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by AP endonuclease creating a nick with 3' hydroxyl and 5' reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was introduced at the 3' hydroxyl group. With near UV-light exposure (312nm) of the extract-probe mixture, only six proteins were strongly labeled, including poly (ADP-ribose) polymerase (PARP-1) and the well-known BER participants flap endonuclease (FEN-1), DNA polymerase b (b-pol), and AP endonuclease (APE). The amount of probe crosslinked to PARP-1 was greater than that crosslinked to the other proteins. The specificity of PARP-1 labeling was examined by competition experiments involving various oligonucleotide competitors; competition of labeling by the probe was much greater for the BER intermediates tested than for normal double-stranded DNA. The specificity of PARP-1 labeling also was examined using DNA probes with alternate structures; PARP-1 labeling was stronger with a DNA oligomer representing a BER intermediate than with a molecule representing a nick in double-stranded DNA. These results identifying interaction of PARP-1 with a BER intermediate are discussed in light of PARP-1's role in mammalian BER.

  12. Base Excision Repair in the Mitochondria

    PubMed Central

    Prakash, Aishwarya; Doublié, Sylvie

    2015-01-01

    The 16.5 kb human mitochondrial genome encodes for 13 polypeptides, 22 tRNAs and 2 rRNAs involved in oxidative phosphorylation. Mitochondrial DNA (mtDNA), unlike its nuclear counterpart, is not packaged into nucleosomes and is more prone to the adverse effects of reactive oxygen species (ROS) generated during oxidative phosphorylation. The past few decades have witnessed an increase in the number of proteins observed to translocate to the mitochondria for the purposes of mitochondrial genome maintenance. The mtDNA damage produced by ROS, if not properly repaired, leads to instability and can ultimately manifest in mitochondrial dysfunction and disease. The base excision repair (BER) pathway is employed for the removal and consequently the repair of deaminated, oxidized, and alkylated DNA bases. Specialized enzymes called DNA glycosylases, which locate and cleave the damaged base, catalyze the first step of this highly coordinated repair pathway. This review focuses on members of the four human BER DNA glycosylase superfamilies and their subcellular localization in the mitochondria and/or the nucleus, as well as summarizes their structural features, biochemical properties, and functional role in the excision of damaged bases. PMID:25754732

  13. Base excision repair: a critical player in many games.

    PubMed

    Wallace, Susan S

    2014-07-01

    This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person's view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field.

  14. Base excision repair: A critical player in many games

    PubMed Central

    Wallace, Susan S.

    2014-01-01

    This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person’s view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field. PMID:24780558

  15. Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair.

    PubMed

    Zhu, Chenxu; Lu, Lining; Zhang, Jun; Yue, Zongwei; Song, Jinghui; Zong, Shuai; Liu, Menghao; Stovicek, Olivia; Gao, Yi Qin; Yi, Chengqi

    2016-07-12

    NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction. PMID:27354518

  16. Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair.

    PubMed

    Zhu, Chenxu; Lu, Lining; Zhang, Jun; Yue, Zongwei; Song, Jinghui; Zong, Shuai; Liu, Menghao; Stovicek, Olivia; Gao, Yi Qin; Yi, Chengqi

    2016-07-12

    NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.

  17. Purification of PCNA as a nucleotide excision repair protein

    PubMed Central

    Nichols, Anne F.; Sancar, Aziz

    1992-01-01

    Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunobloting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases δ and ε. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically. Images PMID:1352873

  18. Base Excision Repair of Oxidative DNA Damage

    PubMed Central

    David, Sheila S.; O’Shea, Valerie L.; Kundu, Sucharita

    2010-01-01

    Base excision repair plays an important role in preventing mutations associated with the common product of oxidative damage, 8-oxoguanine. Recent structural studies have shown that 8-oxoguanine glycosylases use an intricate series of steps to efficiently search and locate 8-oxoguanine lesions within the multitude of undamaged bases. The importance of prevention of mutations associated with 8-oxoguanine has also been illustrated by direct connections between defects in the BER glycosylase MUTYH and colorectal cancer. In addition, the properties of other guanine oxidation products and the BER glycosylases that remove them are being uncovered. This work is providing surprising and intriguing new insights into the process of base excision repair. PMID:17581577

  19. DNA excision repair: where do all the dimers go?

    PubMed

    Kemp, Michael G; Sancar, Aziz

    2012-08-15

    Exposure of cells to UV light from the sun causes the formation of pyrimidine dimers in DNA that have the potential to lead to mutation and cancer. In humans, pyrimidine dimers are removed from the genome in the form of ~30 nt-long oligomers by concerted dual incisions. Though nearly 50 y of excision repair research has uncovered many details of UV photoproduct damage recognition and removal, the fate of the excised oligonucleotides and, in particular, the ultimate fate of the chemically very stable pyrimidine dimers remain unknown. Physiologically relevant UV doses introduce hundreds of thousands of pyrimidine dimers in diploid human cells, which are excised from the genome within ~24 h. Once removed from the genome, "where do all the dimers go?" In a recent study we addressed this question. Although our study did not determine the fate of the dimer itself, it revealed that the excised ~30-mer is released from the duplex in a tight complex with the transcription/repair factor TFIIH. This finding combined with recent reports that base and oligonucleotide products of the base and double-strand break repair pathways also make stable complexes with the cognate repair enzymes, and that these complexes activate the MAP kinase and checkpoint signaling pathways, respectively, raises the possibility that TFIIH-30-mer excision complexes may play a role in signaling reactions in response to UV damage.

  20. Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

    PubMed

    Ma, Huaxian; Wang, Jianling; Abdel-Rahman, Sherif Z; Boor, Paul J; Khan, M Firoze

    2013-03-15

    Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of

  1. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    SciTech Connect

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  2. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

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

  3. Variation in Base Excision Repair Capacity

    PubMed Central

    Wilson, David M.; Kim, Daemyung; Berquist, Brian R.; Sigurdson, Alice J.

    2010-01-01

    The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays. PMID:21167187

  4. Nucleotide excision repair of DNA: The very early history.

    PubMed

    Friedberg, Errol C

    2011-07-15

    This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair.

  5. Nucleotide Excision Repair in Caenorhabditis elegans

    PubMed Central

    Lans, Hannes; Vermeulen, Wim

    2011-01-01

    Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage. PMID:22091407

  6. Base excision repair capacity in informing healthspan

    PubMed Central

    Brenerman, Boris M.; Illuzzi, Jennifer L.; Wilson, David M.

    2014-01-01

    Base excision repair (BER) is a frontline defense mechanism for dealing with many common forms of endogenous DNA damage, several of which can drive mutagenic or cell death outcomes. The pathway engages proteins such as glycosylases, abasic endonucleases, polymerases and ligases to remove substrate modifications from DNA and restore the genome back to its original state. Inherited mutations in genes related to BER can give rise to disorders involving cancer, immunodeficiency and neurodegeneration. Studies employing genetically defined heterozygous (haploinsufficient) mouse models indicate that partial reduction in BER capacity can increase vulnerability to both spontaneous and exposure-dependent pathologies. In humans, measurement of BER variation has been imperfect to this point, yet tools to assess BER in epidemiological surveys are steadily evolving. We provide herein an overview of the BER pathway and discuss the current efforts toward defining the relationship of BER defects with disease susceptibility. PMID:25355293

  7. Mammalian Transcription-Coupled Excision Repair

    PubMed Central

    Vermeulen, Wim; Fousteri, Maria

    2013-01-01

    Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients’ death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process. PMID:23906714

  8. Nucleosomes determine their own patch size in base excision repair.

    PubMed

    Meas, Rithy; Smerdon, Michael J

    2016-01-01

    Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2-12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in 'designed' nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863

  9. Nucleosomes determine their own patch size in base excision repair

    PubMed Central

    Meas, Rithy; Smerdon, Michael J.

    2016-01-01

    Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in ‘designed’ nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863

  10. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    PubMed Central

    Ishino, Sonoko; Connolly, Bernard A.

    2016-01-01

    In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations. PMID:27721668

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

    PubMed

    Hubank, M; Mayne, L

    1994-08-12

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

  12. Inhibition of excision-repair of ultraviolet damage in human cells by exposure to methyl methanesulfonate.

    PubMed

    Park, S D; Choi, K H; Hong, S W; Cleaver, J E

    1981-07-01

    Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves. PMID:7196494

  13. Evidence implying DNA polymerase beta function in excision repair.

    PubMed

    Siedlecki, J A; Szyszko, J; Pietrzykowska, I; Zmudzka, B

    1980-01-25

    Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, being, as expected, 40 times higher than for T-DNA. Likewise, the differences in the yield of the s.s.act.DNA or s.s.BU-DNA replication between both enzymes were negligible. In contrast, damaged native DNA was 6 - 30 times more extensively replicated by DNA polymerase beta than alpha. We propose that this is due to the greater ability of DNA polymerase beta compared with alpha to replicate single-stranded gaps, the presence of which is more likely in damaged BU-DNA than in T-DNA and act.DNA.

  14. Nucleotide excision repair by dual incisions in plants

    PubMed Central

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

    2016-01-01

    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. PMID:27071131

  15. 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. PMID:27071131

  16. Premature aging and cancer in nucleotide excision repair-disorders

    PubMed Central

    Diderich, K.; Alanazi, M.; Hoeijmakers, J.H.J.

    2014-01-01

    During past decades the major impact of DNA damage on cancer as ‘disease of the genes’ has become abundantly apparent. In addition to cancer recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems not only protect against cancer but equally against too fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in man. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging. PMID:21680258

  17. Base excision repair and the role of MUTYH

    PubMed Central

    Kairupan, Carla; Scott, Rodney J

    2007-01-01

    The correction of exogenous and endogenous environmental insult to DNA involves a series of DNA repair mechanisms that reduce the likelihood of mutation accumulation and hence an increased probability of tumour development. The mechanisms underlying the process of base excision repair are relatively well understood and are placed in context with how deterioration of this process is associated with an increased risk of malignancy. PMID:19725997

  18. Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

    PubMed

    Guintini, Laetitia; Charton, Romain; Peyresaubes, François; Thoma, Fritz; Conconi, Antonio

    2015-12-01

    The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

  19. DNA glycosylases in the base excision repair of DNA.

    PubMed Central

    Krokan, H E; Standal, R; Slupphaug, G

    1997-01-01

    A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the N-glycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic/apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3' to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-dimer glycosylase, the enzyme gains access to the target base by flipping out an adenine opposite to the dimer. A conserved helix-hairpin-helix motif and an invariant Asp residue are found in the active sites of more than 20 monofunctional and bifunctional DNA glycosylases. In bifunctional DNA glycosylases, the conserved Asp is thought to deprotonate a conserved Lys, forming an amine nucleophile. The nucleophile forms a covalent intermediate (Schiff base) with the deoxyribose anomeric carbon and expels the base. Deoxyribose subsequently undergoes several transformations, resulting in strand cleavage and regeneration of the free enzyme. The catalytic mechanism of monofunctional glycosylases does not involve covalent intermediates. Instead the conserved Asp residue may activate a water molecule which acts as the attacking nucleophile. PMID:9224623

  20. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  1. HMGB1 is a cofactor in mammalian base excision repair.

    PubMed

    Prasad, Rajendra; Liu, Yuan; Deterding, Leesa J; Poltoratsky, Vladimir P; Kedar, Padmini S; Horton, Julie K; Kanno, Shin-Ichiro; Asagoshi, Kenjiro; Hou, Esther W; Khodyreva, Svetlana N; Lavrik, Olga I; Tomer, Kenneth B; Yasui, Akira; Wilson, Samuel H

    2007-09-01

    Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells. PMID:17803946

  2. A molecular beacon assay for measuring base excision repair activities.

    PubMed

    Maksimenko, Andrei; Ishchenko, Alexander A; Sanz, Guenhaël; Laval, Jacques; Elder, Rhoderick H; Saparbaev, Murat K

    2004-06-18

    The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.

  3. Nucleotide excision repair: new tricks with old bricks.

    PubMed

    Kamileri, Irene; Karakasilioti, Ismene; Garinis, George A

    2012-11-01

    Nucleotide excision repair (NER) is a major DNA repair pathway that ensures that the genome remains functionally intact and is faithfully transmitted to progeny. However, defects in NER lead, in addition to cancer and aging, to developmental abnormalities whose clinical heterogeneity and varying severity cannot be fully explained by the DNA repair deficiencies. Recent work has revealed that proteins in NER play distinct roles, including some that go well beyond DNA repair. NER factors are components of protein complexes known to be involved in nucleosome remodeling, histone ubiquitination, and transcriptional activation of genes involved in nuclear receptor signaling, stem cell reprogramming, and postnatal mammalian growth. Together, these findings add new pieces to the puzzle for understanding NER and the relevance of NER defects in development and disease.

  4. Dynamic control of strand excision during human DNA mismatch repair.

    PubMed

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  5. Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture).

    PubMed

    Sancar, Aziz

    2016-07-18

    Ultraviolet light damages DNA by converting two adjacent thymines into a thymine dimer which is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired by photolyase and the nucleotide excision repair system in E. coli by nucleotide excision repair in humans. The work leading to these results is presented by Aziz Sancar in his Nobel Lecture. PMID:27337655

  6. Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture).

    PubMed

    Sancar, Aziz

    2016-07-18

    Ultraviolet light damages DNA by converting two adjacent thymines into a thymine dimer which is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired by photolyase and the nucleotide excision repair system in E. coli by nucleotide excision repair in humans. The work leading to these results is presented by Aziz Sancar in his Nobel Lecture.

  7. Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair

    PubMed Central

    Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni

    2016-01-01

    Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111

  8. E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair

    PubMed Central

    Vélez-Cruz, Renier; Johnson, David G.

    2012-01-01

    Many of the biochemical details of nucleotide excision repair (NER) have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER. PMID:23202967

  9. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    PubMed Central

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  10. Base excision repair in the mammalian brain: implication for age related neurodegeneration.

    PubMed

    Sykora, Peter; Wilson, David M; Bohr, Vilhelm A

    2013-10-01

    The repair of damaged DNA is essential to maintain longevity of an organism. The brain is a matrix of different neural cell types including proliferative astrocytes and post-mitotic neurons. Post-mitotic DNA repair is a version of proliferative DNA repair, with a reduced number of available pathways and most of these attenuated. Base excision repair (BER) is one pathway that remains robust in neurons; it is this pathway that resolves the damage due to oxidative stress. This oxidative damage is an unavoidable byproduct of respiration, and considering the high metabolic activity of neurons this type of damage is particularly pertinent in the brain. The accumulation of oxidative DNA damage over time is a central aspect of the theory of aging and repair of such chronic damage is of the highest importance. We review research conducted in BER mouse models to clarify the role of this pathway in the neural system. The requirement for BER in proliferating cells also correlates with high levels of many of the BER enzymes in neurogenesis after DNA damage. However, the pathway is also necessary for normal neural maintenance as larger infarct volumes after ischemic stroke are seen in some glycosylase deficient animals. Further, the requirement for DNA polymerase β in post-mitotic BER is potentially more important than in proliferating cells due to reduced levels of replicative polymerases. The BER response may have particular relevance for the onset and progression of many neurodegenerative diseases associated with an increase in oxidative stress including Alzheimer's.

  11. Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

    PubMed

    Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

    2016-06-21

    In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

  12. Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

    PubMed

    Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

    2016-06-21

    In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability. PMID:27158700

  13. Base excision repair in Archaea: back to the future in DNA repair.

    PubMed

    Grasso, Stefano; Tell, Gianluca

    2014-09-01

    Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account.

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

    PubMed Central

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

    2014-01-01

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

  15. Regulation of WRN helicase activity in human base excision repair.

    PubMed

    Ahn, Byungchan; Harrigan, Jeanine A; Indig, Fred E; Wilson, David M; Bohr, Vilhelm A

    2004-12-17

    Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.

  16. Coordination of dual incision and repair synthesis in human nucleotide excision repair

    PubMed Central

    Staresincic, Lidija; Fagbemi, Adebanke F; Enzlin, Jacqueline H; Gourdin, Audrey M; Wijgers, Nils; Dunand-Sauthier, Isabelle; Giglia-Mari, Giuseppina; Clarkson, Stuart G; Vermeulen, Wim; Schärer, Orlando D

    2009-01-01

    Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1-XPF and 3′ to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5′ incision by ERCC1-XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity. PMID:19279666

  17. Coordination of dual incision and repair synthesis in human nucleotide excision repair.

    PubMed

    Staresincic, Lidija; Fagbemi, Adebanke F; Enzlin, Jacqueline H; Gourdin, Audrey M; Wijgers, Nils; Dunand-Sauthier, Isabelle; Giglia-Mari, Giuseppina; Clarkson, Stuart G; Vermeulen, Wim; Schärer, Orlando D

    2009-04-22

    Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity. PMID:19279666

  18. Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

    PubMed Central

    Scott, Timothy L.; Rangaswamy, Suganya; Wicker, Christina A.

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. Recent Advances: In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. Critical Issues: The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. Future Directions: Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation. Antioxid. Redox Signal. 20, 708–726. PMID:23901781

  19. The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair

    PubMed Central

    Kelsall, Ian R.; Langenick, Judith; MacKay, Craig; Patel, Ketan J.; Alpi, Arno F.

    2012-01-01

    The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes – UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair. PMID:22615860

  20. Excision repair of bulky lesions in the DNA of mammalian cells

    SciTech Connect

    Setlow, R B; Grist, E

    1980-01-01

    The report examines the process of excision repair of pyrimidine dimers from uv-irradiated and chemically challenged human cells. It is shown by means of a sensitive endonuclease assay that the amount of excision observed depends upon the isotope used to label cells, and that XP heterozygotes are between normals and XPs. (ACR)

  1. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target.

    PubMed

    Fishel, Melissa L; Kelley, Mark R

    2007-01-01

    With our growing understanding of the pathways involved in cell proliferation and signaling, targeted therapies, in the treatment of cancer are entering the clinical arena. New and emerging targets are proteins involved in DNA repair pathways. Inhibition of various proteins in the DNA repair pathways sensitizes cancer cells to DNA damaging agents such as chemotherapy and/or radiation. We study the apurinic endonuclease 1/redox factor-1 (Ape1/Ref-1) and believe that its crucial function in DNA repair and reduction-oxidation or redox signaling make it an excellent target for sensitizing tumor cells to chemotherapy. Ape1/Ref-1 is an essential enzyme in the base excision repair (BER) pathway which is responsible for the repair of DNA caused by oxidative and alkylation damage. As importantly, Ape1/Ref-1 also functions as a redox factor maintaining transcription factors in an active reduced state. Ape1/Ref-1 stimulates the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as AP-1 (Fos/Jun), NFkappaB, HIF-1alpha, CREB, p53 and others. We will discuss what is known regarding the pharmacological targeting of the DNA repair activity, as well as the redox activity of Ape1/Ref-1, and explore the budding clinical utility of inhibition of either of these functions in cancer treatment. A brief discussion of the effect of polymorphisms in its DNA sequence is included because of Ape1/Ref-1's importance to maintenance and integrity of the genome. Experimental modification of Ape1/Ref-1 activity changes the response of cells and of organisms to DNA damaging agents, suggesting that Ape1/Ref-1 may also be a productive target of chemoprevention. In this review, we will provide an overview of Ape1/Ref-1's activities and explore the potential of this protein as a target in cancer treatment as well as its role in chemoprevention.

  2. Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

    SciTech Connect

    Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

    2002-03-01

    Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.

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

    SciTech Connect

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

    1993-10-01

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

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

    PubMed Central

    Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

    2016-01-01

    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. PMID:27058533

  5. 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

  6. Conversions of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes

    SciTech Connect

    Fenech, M.; Neville, S. )

    1992-01-01

    The human lymphocyte micronucleus (MN) assay is relatively insensitive to genotoxic agents that predominantly induce excision-repairable lesions such as adducts and abasic sites. In this study the authors have explored the possibility of using cytosine arabinoside (ARA) to convert excision-repairable DNA lesions to micronuclei (MN) within one cell cycle. The system consisted of human lymphocytes as target cells, the cytokinesis-block (CB) method for identifying cells that had completed one nuclear division only, and X-rays, methylnitrosourea (MNU), and ultraviolet light (UV) as mutagens. With each mutagen they have observed significant increments induced MN in the cultures that had also been treated with ARA during G{sub 1}. These observations suggested that the combined ARA and cytokinesis-block micronucleus (CBMN) method may enhance the detection of exposure to genotoxic agents that predominantly induce excision-repairable lesions.

  7. Role of Deubiquitinating Enzymes in DNA Repair

    PubMed Central

    2015-01-01

    Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling. PMID:26644404

  8. Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA.

    PubMed

    Aboussekhra, A; Wood, R D

    1995-12-01

    During nucleotide excision repair, damaged DNA is incised on both sides of a lesion and an oligomer containing the damage is excised and replaced by repair DNA synthesis. The latter step is accomplished in vitro by proteins that include the DNA polymerase accessory factor PCNA, which binds to DNA ends to initiate repair synthesis. An increased association of PCNA with nuclei occurs after UV irradiation of nonreplicating DNA in normal human fibroblasts, probably following incision of damaged DNA. This property was used to detect the catalysis of nucleotide excision repair incisions in damaged DNA in vivo, by immunostaining of quiescent human fibroblasts with the widely available PC10 antibody. We summarize here a comprehensive survey of PCNA immunostaining in repair-defective xeroderma pigmentosum (XP) cells in comparison to normal cells. XP-A and XP-G cells were completely defective in staining for PCNA 30 min after UV irradiation. This strongly suggests that XPA and XPG proteins are absolutely required in cells before any incisions can be formed in damaged DNA. XP-B, XP-C, XP-D, and XP-F cells showed an intermediate level of staining for PCNA after UV irradiation, indicative of partial incision capacity in those cells. UV-irradiated XP-E and XP-V cells showed normal PCNA immunostaining levels, consistent with evidence that the corresponding factors are not essential for the incision step of repair. The results provide further evidence for the involvement of PCNA in the repair process in vivo and give an alternative to traditional approaches for measurement of nucleotide excision repair capability. PMID:7493631

  9. POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

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

    SciTech Connect

    Hartman, P.S.; Hevelone, J.; Dwarakanath, V.; Mitchell, D.L. )

    1989-06-01

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

  11. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

    SciTech Connect

    Sykora, Peter; Snow, Elizabeth T.

    2008-05-01

    Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase {beta} (Pol {beta}) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol {beta} and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 {mu}M. However, at lower doses Pol {beta} mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol {beta} was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis.

  12. Modeling the induced mutation process in bacterial cells with defects in excision repair system

    NASA Astrophysics Data System (ADS)

    Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2015-12-01

    A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.

  13. Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins

    PubMed Central

    Poletto, Mattia; Lirussi, Lisa; Wilson, David M.; Tell, Gianluca

    2014-01-01

    Nucleophosmin (NPM1) is a multifunctional protein that controls cell growth and genome stability via a mechanism that involves nucleolar–cytoplasmic shuttling. It is clear that NPM1 also contributes to the DNA damage response, yet its exact function is poorly understood. We recently linked NPM1 expression to the functional activation of the major abasic endonuclease in mammalian base excision repair (BER), apurinic/apyrimidinic endonuclease 1 (APE1). Here we unveil a novel role for NPM1 as a modulator of the whole BER pathway by 1) controlling BER protein levels, 2) regulating total BER capacity, and 3) modulating the nucleolar localization of several BER enzymes. We find that cell treatment with the genotoxin cisplatin leads to concurrent relocalization of NPM1 and BER components from nucleoli to the nucleoplasm, and cellular experiments targeting APE1 suggest a role for the redistribution of nucleolar BER factors in determining cisplatin toxicity. Finally, based on the use of APE1 as a representative protein of the BER pathway, our data suggest a function for BER proteins in the regulation of ribogenesis. PMID:24648491

  14. Nucleotide excision repair and response and survival to chemotherapy in colorectal cancer patients.

    PubMed

    Kap, Elisabeth J; Popanda, Odilia; Chang-Claude, Jenny

    2016-05-01

    Several new chemotherapeutic agents have become available for the treatment of colorectal cancer, which has led to increased complexity in treatment planning. Treatment decision making for individual patients could be facilitated if guided by predictive and prognostic markers. As most cytotoxic drugs induce DNA damage, the DNA damage repair pathways hold potential for yielding such biomarkers. Here, we review the current evidence of a possible involvement of the nucleotide excision repair pathway in the efficacy of chemotherapeutic agents used in the treatment of colorectal cancer. Although a large number of studies have been conducted, they are generally of moderate size and heterogeneous in design. Up to date no firm conclusions can be drawn to translate these results into the clinic. We recommend further comprehensive investigations of the nucleotide excision repair pathway in large patient studies that include both discovery and validation cohorts. PMID:27183147

  15. The impact of base excision DNA repair in age-related neurodegenerative diseases.

    PubMed

    Leandro, Giovana S; Sykora, Peter; Bohr, Vilhelm A

    2015-06-01

    The aging process and several age-related neurodegenerative disorders have been linked to elevated levels of DNA damage induced by ROS and deficiency in DNA repair mechanisms. DNA damage induced by ROS is a byproduct of cellular respiration and accumulation of damage over time, is a fundamental aspect of a main theory of aging. Mitochondria have a pivotal role in generating cellular oxidative stress, and mitochondrial dysfunction has been associated with several diseases. DNA base excision repair is considered the major pathway for repair of oxidized bases in DNA both in the nuclei and in mitochondria, and in neurons this mechanism is particularly important because non-diving cells have limited back-up DNA repair mechanisms. An association between elevated oxidative stress and a decrease in BER is strongly related to the aging process and has special relevance in age-related neurodegenerative diseases. Here, we review the role of DNA repair in aging, focusing on the implications of the DNA base excision repair pathways and how alterations in expression of these DNA repair proteins are related to the aging process and to age-related neurodegenerative diseases.

  16. Comparison of Three Surgical Methods in Treatment of Patients with Pilonidal Sinus: Modified Excision and Repair/Wide Excision/Wide Excision and Flap in RASOUL, OMID and SADR Hospitals( 2004-2007).

    PubMed

    Hosseini, Mostafa; Heidari, Afshin; Jafarnejad, Babak

    2013-10-01

    This study is a comparison between three methods that are frequently used for the surgical treatment of pilonidal disease all over the world: modified excision and repair, wide excision and secondary repair, and wide excision and flap. The first technique is done by our group for the first time, and has not been described previously in the literature. This is an interventional study performed at Omid, Sadr, and Rasoul Akram hospitals on patients who had undergone operation because of pilonidal sinus disease and met the inclusion criteria between 2004 and 2007. Exclusion criteria were (1) acute pilonidal sinus diseases, (2) history of pilonidal sinus surgery, (3) history of systemic diseases (DM, malignancy, etc.), and (4) pilonidal abscess. Essential information was extracted from complete medical archives. Any data not available in files or during follow-up visits (all patients supposed to be followed at least for 1 year) were gathered by a telephone interview. A total of 194 patients met the criteria and had complete archived files. Longer duration of hospital stay was found in the "wide excision and closing with flap" method comparing with two other methods (P < 0.05). Length of incapacity for work was not different between the "wide excision and modified repair" and "wide excision" (P > 0.5) methods, but longer for "wide excision and flap" in comparison with two others (P < 0.05). Healing time was significantly longer in the "wide excision" method in comparison with two other methods (P < 0.05). However, "wide excision and modified repair" method had the least healing time between all above techniques, except for length of leaving the office. All the three recurrences (1.5 %) occurred in the wide excision and flap method (P < 0.05). The frequency of postoperative complications was 2 (3.3 %) in wide excision and modified repair, 15 (18.5 %) in wide excision, and 17 (32.7 %) in wide excision and flap closure; these differences in

  17. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  18. Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

    PubMed

    Moor, Nina A; Vasil'eva, Inna A; Anarbaev, Rashid O; Antson, Alfred A; Lavrik, Olga I

    2015-07-13

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

  19. CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair.

    PubMed

    Ström, Cecilia E; Mortusewicz, Oliver; Finch, David; Parsons, Jason L; Lagerqvist, Anne; Johansson, Fredrik; Schultz, Niklas; Erixon, Klaus; Dianov, Grigory L; Helleday, Thomas

    2011-09-01

    CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.

  20. Assessing tumor mutations to gain insight into base excision repair sequence polymorphisms and smoking in colon cancer

    PubMed Central

    Curtin, Karen; Samowitz, Wade S.; Wolff, Roger K.; Ulrich, Cornelia M.; Caan, Bette J.; Potter, John D.; Slattery, Martha L.

    2009-01-01

    DNA repair enzymes function in major pathways to reverse DNA damage, including base excision repair (BER). Missense polymorphisms in BER repair genes may contribute to differences in DNA repair capacity, specific mutations, and susceptibility to cancer in the presence of exposure to carcinogens such as cigarette smoking. In a study of 1,604 incident colon cancer cases and 1,969 matched population-based controls genotyped for BER variants OGG1 (S326C) and XRCC1 (R194W, R280H, and R399Q), we found no associations with colon cancer overall. However, a two-fold increased risk of BRAF V600E tumor mutation was observed in current and former cigarette smokers homozygous for the OGG1 polymorphism (OR= 2.2, 95%CI 1.02-4.9, recessive model); similar associations were not observed for microsatellite instability, CpG Island Methylator Phenotype, KRAS2 mutations, or TP53 mutations. The XRCC1 R194W polymorphism was associated with a modest increased risk of TP53 tumor mutations in those who regularly smoked cigarettes (OR=1.4, 95%CI 1.02-1.9). These findings point to the importance of studying tumor mutations when examining DNA repair polymorphisms and cigarette smoke exposure to identify potentially relevant associations with colorectal cancer. PMID:19959686

  1. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

    PubMed Central

    Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter

    2016-01-01

    Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078

  2. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers.

    PubMed

    Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter

    2016-01-01

    Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078

  3. Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond.

    PubMed

    Compe, Emmanuel; Egly, Jean-Marc

    2016-06-01

    Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies. PMID:27294439

  4. FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

    PubMed

    Charles Richard, John Lalith; Shukla, Manu Shubhdarshan; Menoni, Hervé; Ouararhni, Khalid; Lone, Imtiaz Nisar; Roulland, Yohan; Papin, Christophe; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-07-01

    FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

  5. FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

    PubMed

    Charles Richard, John Lalith; Shukla, Manu Shubhdarshan; Menoni, Hervé; Ouararhni, Khalid; Lone, Imtiaz Nisar; Roulland, Yohan; Papin, Christophe; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-07-01

    FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129

  6. FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC

    PubMed Central

    Ouararhni, Khalid; Roulland, Yohan; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-01-01

    FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129

  7. Gut Microbiota Imbalance and Base Excision Repair Dynamics in Colon Cancer

    PubMed Central

    Ray, Debolina; Kidane, Dawit

    2016-01-01

    Gut microbiota are required for host nutrition, energy balance, and regulating immune homeostasis, however, in some cases, this mutually beneficial relationship becomes twisted (dysbiosis), and the gut flora can incite pathological disorders including colon cancer. Microbial dysbiosis promotes the release of bacterial genotoxins, metabolites, and causes chronic inflammation, which promote oxidative DNA damage. Oxidized DNA base lesions are removed by base excision repair (BER), however, the role of this altered function of BER, as well as microbiota-mediated genomic instability and colon cancer development, is still poorly understood. In this review article, we will discuss how dysbiotic microbiota induce DNA damage, its impact on base excision repair capacity, the potential link of host BER gene polymorphism, and the risk of dysbiotic microbiota mediated genomic instability and colon cancer. PMID:27471558

  8. Exploiting Base Excision Repair to Improve Therapeutic Approaches for Pancreatic Cancer

    PubMed Central

    Sharbeen, George; McCarroll, Joshua; Goldstein, David; Phillips, Phoebe A.

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDA) is a highly chemoresistant and metastatic disease with a dismal 5-year survival rate of 6%. More effective therapeutic targets and approaches are urgently needed to tackle this devastating disease. The base excision repair (BER) pathway has been identified as a predictor of therapeutic response, prognostic factor, and therapeutic target in a variety of cancers. This review will discuss our current understanding of BER in PDA and its potential to improve PDA treatment. PMID:25988138

  9. Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae

    PubMed Central

    Stephenson, Adrienne P.; Mazu, Tryphon K.; Miles, Jana S.; Freeman, Miles D.; Reams, R. Renee; Flores-Rozas, Hernan

    2013-01-01

    Manganese (Mn) is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese. PMID:24282812

  10. Main factors providing specificity of repair enzymes.

    PubMed

    Nevinsky, G A

    2011-01-01

    Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10(-4)-10(-8) M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links "covered" by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10(-1)-10(-2) M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10-15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate (k(cat)) increases by 4-8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes. PMID:21568843

  11. The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae.

    PubMed

    Meas, Rithy; Smerdon, Michael J; Wyrick, John J

    2015-05-26

    Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair.

  12. Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Huston, Donald; Kuban, Wojciech; Liu, Lili; Van Houten, Bennett; Woodgate, Roger

    2013-01-01

    Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the

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

    PubMed Central

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

    2014-01-01

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

  14. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    SciTech Connect

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  15. Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

    PubMed Central

    Gaillard, P H; Moggs, J G; Roche, D M; Quivy, J P; Becker, P B; Wood, R D; Almouzni, G

    1997-01-01

    To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited. PMID:9321407

  16. Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair

    PubMed Central

    Latimer, Jean J.; Kelly, Crystal M.

    2016-01-01

    The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. PMID:24623250

  17. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies.

    PubMed

    Shukla, Ankita; Moussa, Ahmed; Singh, Tiratha Raj

    2016-01-01

    DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40-60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels.

  18. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies.

    PubMed

    Shukla, Ankita; Moussa, Ahmed; Singh, Tiratha Raj

    2016-01-01

    DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40-60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels. PMID:27276067

  19. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies

    PubMed Central

    Shukla, Ankita; Singh, Tiratha Raj

    2016-01-01

    DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40–60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels. PMID:27276067

  20. Direct and indirect roles of RECQL4 in modulating base excision repair capacity.

    PubMed

    Schurman, Shepherd H; Hedayati, Mohammad; Wang, ZhengMing; Singh, Dharmendra K; Speina, Elzbieta; Zhang, Yongqing; Becker, Kevin; Macris, Margaret; Sung, Patrick; Wilson, David M; Croteau, Deborah L; Bohr, Vilhelm A

    2009-09-15

    RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H(2)O(2)-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H(2)O(2)-induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H(2)O(2), RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase beta, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity.

  1. Nucleotide excision repair is impaired by binding of transcription factors to DNA.

    PubMed

    Sabarinathan, Radhakrishnan; Mularoni, Loris; Deu-Pons, Jordi; Gonzalez-Perez, Abel; López-Bigas, Núria

    2016-04-14

    Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations.

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

    PubMed

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

    2015-01-01

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

  3. A Case of Microangiopathic Hemolytic Anemia after Myxoma Excision and Mitral Valve Repair Presenting as Hemolytic Uremic Syndrome

    PubMed Central

    Park, Young Joo; Kim, Sang Pil; Shin, Ho-Jin

    2016-01-01

    Microangiopathic hemolytic anemia occurs in a diverse group of disorders, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and prosthetic cardiac valves. Hemolytic anemia also occurs as a rare complication after mitral valve repair. In this report, we describe a case of microangiopathic hemolytic anemia following myxoma excision and mitral valve repair, which was presented as hemolytic uremic syndrome. PMID:27081450

  4. A Case of Microangiopathic Hemolytic Anemia after Myxoma Excision and Mitral Valve Repair Presenting as Hemolytic Uremic Syndrome.

    PubMed

    Park, Young Joo; Kim, Sang Pil; Shin, Ho-Jin; Choi, Jung Hyun

    2016-03-01

    Microangiopathic hemolytic anemia occurs in a diverse group of disorders, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and prosthetic cardiac valves. Hemolytic anemia also occurs as a rare complication after mitral valve repair. In this report, we describe a case of microangiopathic hemolytic anemia following myxoma excision and mitral valve repair, which was presented as hemolytic uremic syndrome. PMID:27081450

  5. UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

    SciTech Connect

    Vijg, J.; Mullaart, E.; Berends, F.; Lohman, P.H.; Knook, D.L.

    1986-12-01

    UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.

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

    PubMed Central

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

    2016-01-01

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

  7. The Friedreich's ataxia protein frataxin modulates DNA base excision repair in prokaryotes and mammals.

    PubMed

    Thierbach, René; Drewes, Gunnar; Fusser, Markus; Voigt, Anja; Kuhlow, Doreen; Blume, Urte; Schulz, Tim J; Reiche, Carina; Glatt, Hansruedi; Epe, Bernd; Steinberg, Pablo; Ristow, Michael

    2010-11-15

    DNA-repair mechanisms enable cells to maintain their genetic information by protecting it from mutations that may cause malignant growth. Recent evidence suggests that specific DNA-repair enzymes contain ISCs (iron-sulfur clusters). The nuclearencoded protein frataxin is essential for the mitochondrial biosynthesis of ISCs. Frataxin deficiency causes a neurodegenerative disorder named Friedreich's ataxia in humans. Various types of cancer occurring at young age are associated with this disease, and hence with frataxin deficiency. Mice carrying a hepatocyte-specific disruption of the frataxin gene develop multiple liver tumours for unresolved reasons. In the present study, we show that frataxin deficiency in murine liver is associated with increased basal levels of oxidative DNA base damage. Accordingly, eukaryotic V79 fibroblasts overexpressing human frataxin show decreased basal levels of these modifications, while prokaryotic Salmonella enterica serotype Typhimurium TA104 strains transformed with human frataxin show decreased mutation rates. The repair rates of oxidative DNA base modifications in V79 cells overexpressing frataxin were significantly higher than in control cells. Lastly, cleavage activity related to the ISC-independent repair enzyme 8-oxoguanine glycosylase was found to be unaltered by frataxin overexpression. These findings indicate that frataxin modulates DNA-repair mechanisms probably due to its impact on ISC-dependent repair proteins, linking mitochondrial dysfunction to DNA repair and tumour initiation.

  8. Investigation of bacterial nucleotide excision repair using single-molecule techniques

    PubMed Central

    Van Houten, Bennett; Kad, Neil

    2016-01-01

    Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER. PMID:24472181

  9. Vertebrate POLQ and POLβ Cooperate in Base Excision Repair of Oxidative DNA Damage

    PubMed Central

    Yoshimura, Michio; Kohzaki, Masaoki; Nakamura, Jun; Asagoshi, Kenjiro; Sonoda, Eiichiro; Hou, Esther; Prasad, Rajendra; Wilson, Samuel H.; Tano, Keizo; Yasui, Akira; Lan, Li; Seki, Mineaki; Wood, Richard D.; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hochegger, Helfrid; Okada, Takashi; Hiraoka, Masahiro; Takeda, Shunichi

    2007-01-01

    Summary Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H2O2, but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLβ. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLβ, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLβ share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER. PMID:17018297

  10. The role of DNA base excision repair in brain homeostasis and disease.

    PubMed

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah; Bohr, Vilhelm A

    2015-08-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of proteins required for BER or proteins that regulate BER have been consistently associated with neurological dysfunction and disease in humans. Recent studies suggest that DNA lesions in the nuclear and mitochondrial compartments and the cellular response to those lesions have a profound effect on cellular energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease.

  11. The role of DNA base excision repair in brain homeostasis and disease.

    PubMed

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah; Bohr, Vilhelm A

    2015-08-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of proteins required for BER or proteins that regulate BER have been consistently associated with neurological dysfunction and disease in humans. Recent studies suggest that DNA lesions in the nuclear and mitochondrial compartments and the cellular response to those lesions have a profound effect on cellular energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease. PMID:26002197

  12. SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

    PubMed Central

    van Cuijk, Loes; van Belle, Gijsbert J.; Turkyilmaz, Yasemin; Poulsen, Sara L.; Janssens, Roel C.; Theil, Arjan F.; Sabatella, Mariangela; Lans, Hannes; Mailand, Niels; Houtsmuller, Adriaan B.; Vermeulen, Wim; Marteijn, Jurgen A.

    2015-01-01

    XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4DDB2 ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER. PMID:26151477

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

    SciTech Connect

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

    1994-10-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Liu, X; Smerdon, M J

    2000-08-01

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

  16. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    PubMed Central

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  17. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    PubMed

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.

  18. Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice

    PubMed Central

    Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja; Gregersen, Ida; Dahl, Tuva B.; Suganthan, Rajikala; Segers, Filip M.; Østlie, Ingunn; Otten, Jeroen J. T.; Luna, Luisa; Ketelhuth, Daniel F. J.; Lundberg, Anna M.; Neurauter, Christine G.; Hildrestrand, Gunn; Skjelland, Mona; Bjørndal, Bodil; Svardal, Asbjørn M.; Iversen, Per O.; Hedin, Ulf; Nygård, Ståle; Olstad, Ole K.; Krohg-Sørensen, Kirsten; Slupphaug, Geir; Eide, Lars; Kuśnierczyk, Anna; Folkersen, Lasse; Ueland, Thor; Berge, Rolf K.; Hansson, Göran K.; Biessen, Erik A. L.; Halvorsen, Bente; Bjørås, Magnar; Aukrust, Pål

    2016-01-01

    Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe−/−Neil3−/− mice on high-fat diet showed accelerated plaque formation as compared to Apoe−/− mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe−/−Neil3−/− mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage. PMID:27328939

  19. Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice.

    PubMed

    Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja; Gregersen, Ida; Dahl, Tuva B; Suganthan, Rajikala; Segers, Filip M; Østlie, Ingunn; Otten, Jeroen J T; Luna, Luisa; Ketelhuth, Daniel F J; Lundberg, Anna M; Neurauter, Christine G; Hildrestrand, Gunn; Skjelland, Mona; Bjørndal, Bodil; Svardal, Asbjørn M; Iversen, Per O; Hedin, Ulf; Nygård, Ståle; Olstad, Ole K; Krohg-Sørensen, Kirsten; Slupphaug, Geir; Eide, Lars; Kuśnierczyk, Anna; Folkersen, Lasse; Ueland, Thor; Berge, Rolf K; Hansson, Göran K; Biessen, Erik A L; Halvorsen, Bente; Bjørås, Magnar; Aukrust, Pål

    2016-01-01

    Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe(-/-)Neil3(-/-) mice on high-fat diet showed accelerated plaque formation as compared to Apoe(-/-) mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe(-/-)Neil3(-/-) mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage. PMID:27328939

  20. The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

    PubMed

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-11-01

    Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.

  1. XRCC1 and base excision repair balance in response to nitric oxide.

    PubMed

    Mutamba, James T; Svilar, David; Prasongtanakij, Somsak; Wang, Xiao-Hong; Lin, Ying-Chih; Dedon, Peter C; Sobol, Robert W; Engelward, Bevin P

    2011-12-10

    Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO(-)) and nitric oxide (NO), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO(-) donor, SIN-1, and to gaseous NO. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NO and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NO-induced DNA lesions: 1,N(6)-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NO-induced DNA damage. These results show that AAG is a key glycosylase for BER of NO-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk.

  2. Nucleotide Excision DNA Repair is Associated with Age-Related Vascular Dysfunction

    PubMed Central

    Durik, Matej; Kavousi, Maryam; van der Pluijm, Ingrid; Isaacs, Aaron; Cheng, Caroline; Verdonk, Koen; Loot, Annemarieke E.; Oeseburg, Hisko; Musterd-Bhaggoe, Usha; Leijten, Frank; van Veghel, Richard; de Vries, Rene; Rudez, Goran; Brandt, Renata; Ridwan, Yanto R.; van Deel, Elza D.; de Boer, Martine; Tempel, Dennie; Fleming, Ingrid; Mitchell, Gary F.; Verwoert, Germaine C.; Tarasov, Kirill V.; Uitterlinden, Andre G.; Hofman, Albert; Duckers, Henricus J.; van Duijn, Cornelia M.; Oostra, Ben A.; Witteman, Jacqueline C.M.; Duncker, Dirk J.; Danser, A.H. Jan; Hoeijmakers, Jan H.; Roks, Anton J.M.

    2012-01-01

    Background Vascular dysfunction in atherosclerosis and diabetes, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability due to the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function as compared to wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness and elevated blood pressure at very young age. The vasodilator dysfunction was due to decreased endothelial eNOS levels as well as impaired smooth muscle cell function, which involved phosphodiesterase (PDE) activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single nucleotide polymorphisms (SNPs) of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium, and found a significant association of a SNP (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans, but with an accelerated progression, as compared to wild type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease. PMID:22705887

  3. Dynamic Interaction of TTDA with TFIIH Is Stabilized by Nucleotide Excision Repair in Living Cells

    PubMed Central

    Theil, Arjan F; Mari, Pierre-Olivier; Hoogstraten, Deborah; Ng, Jessica M. Y; Dinant, Christoffel; Hoeijmakers, Jan H. J

    2006-01-01

    Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER. PMID:16669699

  4. Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

    PubMed

    Page, Melissa M; Salmon, Adam B; Leiser, Scott F; Robb, Ellen L; Brown, Melanie F; Miller, Richard A; Stuart, Jeffrey A

    2009-04-15

    Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.

  5. Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

    PubMed

    Page, Melissa M; Salmon, Adam B; Leiser, Scott F; Robb, Ellen L; Brown, Melanie F; Miller, Richard A; Stuart, Jeffrey A

    2009-04-15

    Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts. PMID:19439226

  6. Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair.

    PubMed

    Panigrahi, Sunil K; Hopkins, Kevin M; Lieberman, Howard B

    2015-05-19

    RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

  7. The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks.

    PubMed

    Bourret, Travis J; Lawrence, Kevin A; Shaw, Jeff A; Lin, Tao; Norris, Steven J; Gherardini, Frank C

    2016-01-01

    The Lyme disease spirochete Borrelia burgdorferi encounters a wide range of environmental conditions as it cycles between ticks of the genus Ixodes and its various mammalian hosts. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent antimicrobial molecules generated during the innate immune response to infection, however, it is unclear whether ROS and RNS pose a significant challenge to B. burgdorferi in vivo. In this study, we screened a library of B. burgdorferi strains with mutations in DNA repair genes for increased susceptibility to ROS or RNS in vitro. Strains with mutations in the methyl-directed mismatch repair gene mutS1 are hypersensitive to killing by ROS, while strains lacking the nucleotide excision repair (NER) gene uvrB show increased susceptibility to both ROS and RNS. Therefore, mutS1-deficient and uvrB-deficient strains were compared for their ability to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks to help identify sites of oxidative and nitrosative stresses encountered by B. burgdorferi in vivo. Both mutS1 and uvrB were dispensable for infection of mice, while uvrB promoted the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggest that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks. PMID:27656169

  8. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

    PubMed Central

    Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M.; Dianov, Grigory L.

    2015-01-01

    Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell ‘hallmarks’. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells. PMID:25800737

  9. Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae.

    PubMed

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J

    2014-04-01

    The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

  10. Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26.

    PubMed

    Li, Shisheng

    2015-12-01

    Transcription coupled nucleotide excision repair (TC-NER) is believed to be triggered by an RNA polymerase stalled at a lesion in the transcribed strand of actively transcribed genes. Rad26, a DNA-dependent ATPase in the family of SWI2/SNF2 chromatin remodeling proteins, plays an important role in TC-NER in Saccharomyces cerevisiae. However, Rad26 is not solely responsible for TC-NER and Rpb9, a nonessential subunit of RNA polymerase II (RNAP II), is largely responsible for Rad26-independent TC-NER. The Rad26-dependent and Rpb9-dependent TC-NER have different efficiencies in genes with different transcription levels and in different regions of a gene. Rad26 becomes entirely or partially dispensable for TC-NER in the absence of Rpb4, another nonessential subunit of RNAP II, or a number of transcription elongation factors (Spt4, Spt5 and the RNAP II associated factor complex). Rad26 may not be a true transcription-repair coupling factor that recruits the repair machinery to the damaged sites where RNAP II stalls. Rather, Rad26 may facilitate TC-NER indirectly, by antagonizing the action of TC-NER repressors that normally promote transcription elongation. The underlying mechanism of how Rad26 functions in TC-NER remains to be elucidated.

  11. The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans.

    PubMed

    Stergiou, L; Doukoumetzidis, K; Sendoel, A; Hengartner, M O

    2007-06-01

    Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.

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

    PubMed Central

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

    2012-01-01

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

  13. Oxidative DNA damage background estimated by a system model of base excision repair

    SciTech Connect

    Sokhansanj, B A; Wilson, III, D M

    2004-05-13

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

  14. The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks

    PubMed Central

    Bourret, Travis J.; Lawrence, Kevin A.; Shaw, Jeff A.; Lin, Tao; Norris, Steven J.; Gherardini, Frank C.

    2016-01-01

    The Lyme disease spirochete Borrelia burgdorferi encounters a wide range of environmental conditions as it cycles between ticks of the genus Ixodes and its various mammalian hosts. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent antimicrobial molecules generated during the innate immune response to infection, however, it is unclear whether ROS and RNS pose a significant challenge to B. burgdorferi in vivo. In this study, we screened a library of B. burgdorferi strains with mutations in DNA repair genes for increased susceptibility to ROS or RNS in vitro. Strains with mutations in the methyl-directed mismatch repair gene mutS1 are hypersensitive to killing by ROS, while strains lacking the nucleotide excision repair (NER) gene uvrB show increased susceptibility to both ROS and RNS. Therefore, mutS1-deficient and uvrB-deficient strains were compared for their ability to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks to help identify sites of oxidative and nitrosative stresses encountered by B. burgdorferi in vivo. Both mutS1 and uvrB were dispensable for infection of mice, while uvrB promoted the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggest that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks.

  15. The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks

    PubMed Central

    Bourret, Travis J.; Lawrence, Kevin A.; Shaw, Jeff A.; Lin, Tao; Norris, Steven J.; Gherardini, Frank C.

    2016-01-01

    The Lyme disease spirochete Borrelia burgdorferi encounters a wide range of environmental conditions as it cycles between ticks of the genus Ixodes and its various mammalian hosts. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent antimicrobial molecules generated during the innate immune response to infection, however, it is unclear whether ROS and RNS pose a significant challenge to B. burgdorferi in vivo. In this study, we screened a library of B. burgdorferi strains with mutations in DNA repair genes for increased susceptibility to ROS or RNS in vitro. Strains with mutations in the methyl-directed mismatch repair gene mutS1 are hypersensitive to killing by ROS, while strains lacking the nucleotide excision repair (NER) gene uvrB show increased susceptibility to both ROS and RNS. Therefore, mutS1-deficient and uvrB-deficient strains were compared for their ability to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks to help identify sites of oxidative and nitrosative stresses encountered by B. burgdorferi in vivo. Both mutS1 and uvrB were dispensable for infection of mice, while uvrB promoted the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggest that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks. PMID:27656169

  16. CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis

    PubMed Central

    Cazzalini, Ornella; Sommatis, Sabrina; Tillhon, Micol; Dutto, Ilaria; Bachi, Angela; Rapp, Alexander; Nardo, Tiziana; Scovassi, A. Ivana; Necchi, Daniela; Cardoso, M. Cristina; Stivala, Lucia A.; Prosperi, Ennio

    2014-01-01

    The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability. PMID:24939902

  17. CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

    PubMed

    Cazzalini, Ornella; Sommatis, Sabrina; Tillhon, Micol; Dutto, Ilaria; Bachi, Angela; Rapp, Alexander; Nardo, Tiziana; Scovassi, A Ivana; Necchi, Daniela; Cardoso, M Cristina; Stivala, Lucia A; Prosperi, Ennio

    2014-07-01

    The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

  18. In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

    PubMed Central

    Moné, Martijn J.; Bernas, Tytus; Dinant, Christoffel; Goedvree, Feliks A.; Manders, Erik M. M.; Volker, Marcel; Houtsmuller, Adriaan B.; Hoeijmakers, Jan H. J.; Vermeulen, Wim; van Driel, Roel

    2004-01-01

    Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of ≈30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells. PMID:15520397

  19. Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.

    PubMed

    Mirbahai, Leda; Kershaw, Rachael M; Green, Richard M; Hayden, Rachel E; Meldrum, Rosalind A; Hodges, Nikolas J

    2010-02-01

    An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.

  20. Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline.

    PubMed

    Cabelof, Diane C; Yanamadala, Sunitha; Raffoul, Julian J; Guo, ZhongMao; Soofi, Abdulsalam; Heydari, Ahmad R

    2003-03-01

    Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.

  1. UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells

    PubMed Central

    2012-01-01

    Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS. PMID:22621766

  2. UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells.

    PubMed

    Sarasin, Alain

    2012-01-01

    Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS. PMID:22621766

  3. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

    PubMed

    Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

    2015-10-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337

  4. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

    PubMed

    Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

    2015-10-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

  5. Modeling nucleotide excision repair and its impact on UV-induced mutagenesis during SOS-response in bacterial cells.

    PubMed

    Bugay, Aleksandr N; Krasavin, Evgeny A; Parkhomenko, Aleksandr Yu; Vasilyeva, Maria A

    2015-01-01

    A model of the UV-induced mutation process in Escherichia coli bacteria has been developed taking into account the whole sequence of molecular events starting from initial photo-damage and finishing with the fixation of point mutations. The wild-type phenotype bacterial cells are compared with UV-sensitive repair-deficient mutant cells. Attention is mainly paid to excision repair system functioning as regards induced mutagenesis.

  6. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway

    PubMed Central

    Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K.; Gairola, C. Gary; Mellon, Isabel

    2016-01-01

    Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke. PMID:27391141

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

    PubMed

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

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

  8. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

    PubMed

    Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K; Gairola, C Gary; Mellon, Isabel

    2016-01-01

    Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke. PMID:27391141

  9. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

    PubMed

    Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K; Gairola, C Gary; Mellon, Isabel

    2016-01-01

    Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

  10. A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

    PubMed Central

    Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff S.; Tang, Christoph M.

    2013-01-01

    Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. PMID:22296581

  11. Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.

    PubMed

    Berra, Carolina Maria; de Oliveira, Carla Santos; Garcia, Camila Carrião Machado; Rocha, Clarissa Ribeiro Reily; Lerner, Letícia Koch; Lima, Leonardo Carmo de Andrade; Baptista, Maurício da Silva; Menck, Carlos Frederico Martins

    2013-08-01

    The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical

  12. Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution.

    PubMed

    Hu, Jinchuan; Adar, Sheera; Selby, Christopher P; Lieb, Jason D; Sancar, Aziz

    2015-05-01

    We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

  13. Fructan metabolising enzymes in rhizophores of Vernonia herbacea upon excision of aerial organs.

    PubMed

    Asega, Amanda Francine; de Carvalho, Maria Angela Machado

    2004-04-01

    The activities of fructan metabolising enzymes and fructan contents are reported for rhizophores of Vernonia herbacea (Vell.) Rusby induced to sprouting by shoot excision. The activities of fructan exohydrolase (1-FEH), sucrose: sucrose fructosyltransferase (1-SST), fructan: fructan fructosyltransferase (1-FFT) and invertase (INV) and the fructan contents were analysed every 3-4 days for 1 month by colorimetric and chromatographic methods. Sprouting of new shoots started on day 9. 1-FEH activity increased after day 13 and reached its maximum value 20 days after shoot excision. A gradual decrease in 1-SST activity was detected between days 3 and 9. 1-FFT activity exhibited fluctuations throughout the experimental period and a peak of activity for invertase was detected 9 days after shoot excision. Variation in fructan contents in vivo included a decrease until day 13 after which, levels remained practically unchanged. Fructan depolymerization and sprouting are concomitant processes in V. herbacea and can be induced by shoot excision at any phenological phase. 1-FEH and 1-FFT seemed to act in a concerted way to catalyse fructan depolymerization, while 1-SST was inhibited, possibly due to interruption of sucrose supply to rhizophores from the aerial organs.

  14. AP Endonuclease 1 as a Key Enzyme in Repair of Apurinic/Apyrimidinic Sites.

    PubMed

    Dyrkheeva, N S; Lebedeva, N A; Lavrik, O I

    2016-09-01

    Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety. APE1 exhibits 3'-phosphodiesterase, 3'-5'-exonuclease, and 3'-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system. PMID:27682167

  15. Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors.

    PubMed

    Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

    2004-04-30

    In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH. PMID:14981083

  16. Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors*

    PubMed Central

    Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H.; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

    2015-01-01

    In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position −3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position −6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position −19 to +8. XPG and XPF/ERCC1 endo-nucleases then cut the damaged DNA at the limit of the opened structure that was previously “labeled” by the positioning of XPC/HR23B and TFIIH. PMID:14981083

  17. Regulation and Disregulation of Mammalian Nucleotide Excision Repair: a Pathway to Non-germline Breast Carcinogenesis†

    PubMed Central

    Latimer, Jean J.; Majekwana, Vongai J.; Pabón-Padín, Yashira R.; Pimpley, Manasi R.; Grant, Stephen G.

    2015-01-01

    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 NER 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 paper, 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. PMID:25393451

  18. Oxidative damage to RPA limits the nucleotide excision repair capacity of human cells

    PubMed Central

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-01-01

    Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin, interact with UVA radiation to generate reactive oxygen species (ROS) that diminish NER capacity by causing protein damage. The RPA DNA binding protein plays a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself is likely to be an important contributor to skin cancer risk. PMID:26134950

  19. DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

    PubMed

    Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I

    2016-03-01

    Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196

  20. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins.

    PubMed

    Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A; Hart, Anne C

    2016-03-01

    Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work.

  1. Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

    PubMed

    Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa; Akopiants, Konstantin; Zhou, Tong; Yannone, Steven M; Ramsden, Dale A; Hartman, Matthew C T; Povirk, Lawrence F

    2016-05-01

    DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. PMID:27049455

  2. Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

    SciTech Connect

    Wu Mei; Zhang Zunzhen Che Wangjun

    2008-05-01

    Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

  3. Developing an in silico model of the modulation of base excision repair using methoxyamine for more targeted cancer therapeutics.

    PubMed

    Gurkan-Cavusoglu, Evren; Avadhani, Sriya; Liu, Lili; Kinsella, Timothy J; Loparo, Kenneth A

    2013-04-01

    Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair. PMID:23847811

  4. AML outcome: role of nucleotide excision repair polymorphisms in intermediate risk patients

    PubMed Central

    Strom, Sara S; Estey, Elihu H; Outschoorn, Ubaldo Martinez; Guillermo, Garcia-Manero

    2010-01-01

    Purpose Acute Myeloid Leukemia (AML) is frequently associated with genetic abnormalities. Based on pre-treatment cytogenetics, patients are classified into favorable, intermediate and poor subgroups. Cytogenetics predicts treatment outcome for the favorable and poor subgroups but not for the intermediate subgroup. Polymorphisms within the nucleotide excision repair (NER) pathway may lead to inter-individual differences in DNA repair capacity (DRC) which could influence outcome. Methods We studied the role of 6 polymorphisms (ERCC1 Gln504Lys, XPD Lys751Gln, XPC Ala499Val, XPC Lys939Gln, XPG Asp1104His, and CCNH Val270Ala) within NER pathway on overall and disease-free survival among 170 adult de-novo AML patients with intermediate cytogenetics [diploid (n=117); non-diploid (n=53)], treated with induction chemotherapy. Kaplan-Meier methods and Cox proportional hazards models were performed. Results Diploid patients with the XPD AC/CC genotype survived shorter than those with the wild-type (AA) genotype (median survival 22 vs. 40 months, log-rank p = 0.03). Similarly diploid patients with XPC CT/TT genotype survived shorter than those with the wild-type (CC) genotype (median survival 15 vs. 30 months, log-rank p = 0.02). Among diploid patients, after adjusting for clinical and socio-demographic variables, patients carrying both XPD AC/CC and XPC CT/TT had a greater than two-fold increased risk of dying compared to those with the wild-type genotypes (HR=2.49; 95%CI: 1.06–5.85). No significant associations were observed for disease-free survival in AML patients. Conclusion By reduced DRC, this combined genotype may result in greater susceptibility to treatment effects decreasing overall survival. These findings could in the future help in selecting treatment strategies for patients with normal cytogenetics. PMID:20141440

  5. A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine.

    PubMed

    Ponferrada-Marín, María Isabel; Parrilla-Doblas, Jara Teresa; Roldán-Arjona, Teresa; Ariza, Rafael R

    2011-03-01

    DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases. PMID:21036872

  6. RNA interference against transcription elongation factor SII does not support its role in transcription-coupled nucleotide excision repair.

    PubMed

    Mackinnon-Roy, Christine; Stubbert, Lawton J; McKay, Bruce C

    2011-01-10

    RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.

  7. Base excision DNA repair levels in mitochondrial lysates of Alzheimer's disease.

    PubMed

    Canugovi, Chandrika; Shamanna, Raghavendra A; Croteau, Deborah L; Bohr, Vilhelm A

    2014-06-01

    Alzheimer's disease (AD) is a senile dementia with increased incidence in older subjects (age >65 years). One of the earliest markers of AD is oxidative DNA damage. Recently, it has been reported that preclinical AD patient brains show elevated levels of oxidative damage in both nuclear and mitochondrial nucleic acids. Moreover, different oxidative lesions in mitochondrial DNA are between 5- and 10-fold higher than in nuclear DNA in both control and AD postmortem brains. We previously showed that there is a significant loss of base excision repair (BER) components in whole tissue extracts of AD and mild cognitive impairment subjects relative to matched control subjects. However, comprehensive analysis of specific steps in BER levels in mitochondrial extracts of AD patient brains is not available. In this study, we mainly investigated various components of BER in mitochondrial extracts of AD and matched control postmortem brain samples. We found that the 5-hydroxyuracil incision and ligase activities are significantly lower in AD brains, whereas the uracil incision, abasic site cleavage, and deoxyribonucleotide triphosphate incorporation activities are normal in these samples.

  8. Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice.

    PubMed

    van Steeg, H; Mullenders, L H; Vijg, J

    2000-05-30

    Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA(-/-) mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA(-/-) mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA(-/-) mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis. PMID:10838141

  9. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

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

    PubMed Central

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

    2011-01-01

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

  11. The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

    SciTech Connect

    Abbasi, Rashda; Efferth, Thomas; Kuhmann, Christine; Opatz, Till; Hao, Xiaojiang; Popanda, Odilia; Schmezer, Peter

    2012-03-15

    Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy

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

    PubMed

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

    1995-09-14

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

  13. UV-induced endonuclease III-sensitive sites at the mating type loci in Saccharomyces cerevisiae are repaired by nucleotide excision repair: RAD7 and RAD16 are not required for their removal from HML alpha.

    PubMed

    Reed, S H; Boiteux, S; Waters, R

    1996-03-01

    Ultraviolet irradiation of DNA induces cyclobutane pyrimidine dimers (CPDs) 6-4'-(pyrimidine 2'-one) pyrimidines and pyrimidine hydrates. The dimer is the major photoproduct, and is specifically recognized by endonuclease V of phage T4. Pyrimidine hydrates represent a small fraction of the total photoproducts, and are substrates for endonuclease III of Escherichia coli. We used these enzymes to follow the fate of their substrates in the mating type loci of Saccharomyces cerevisiae. In a RAD strain, CPSs in the transcriptionally active MAT alpha locus are preferentially repaired relative to the inactive HML alpha locus, whilst repair of endonuclease III-sensitive sites is not preferential. The rad1, 2, 3 and 4 mutants, which lack factors that are essential for the incision step of nucleotide excision repair (NER), repair neither CPDs nor endonuclease III-sensitive sites, clearly showing that these lesions are repaired by by NER pathway. Previously it had been shown that the products of the RAD7 and RAD16 genes are required for the NER of CPDs from the HML alpha locus. We show that, in the same locus, these gene products are not needed for removal of endonuclease III-sensitive sites by the same mechanism. This indicates that the components required for NER differ depending on either the type of lesion encountered or on the specific location of the lesion within the genome.

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

    PubMed Central

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

    1996-01-01

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

  15. [Effects of partial root excision on the growth, photosynthesis, and antioxidant enzyme activities of maize under salt stress].

    PubMed

    Zhang, Hong; Cui, Li-Na; Meng, Jia-Jia; Zhang, Hai-Yan; Shi, De-Yang; Dong, Shu-Ting; Zhang, Ji-Wang; Liu, Peng

    2012-12-01

    A pot experiment was conducted to study the effects of partial root excision on the growth of two maize cultivars (Zhengdan 958 and Denghai 9) throughout their growth period and the photosynthesis and leaf antioxidant enzyme activities at grain-filling stage under salt stress. Four treatments were installed, i. e., control (no salt), low salt (0.2% NaCl), moderate salt (0.4% NaCl), and high salt (0.6% NaCl). Under low salt stress, the grain yield of Zhengdan 958 and Denghai 9 with partial root excision was increased by 13.1% and 31.4%, respectively, as compared with that of the cultivars with no root excision. At jointing stage, the growth of the cultivars with partial root excision was restrained, the root- and shoot dry masses under the same salt stresses being lesser than those of the cultivars with no root excision, but the growth under the conditions of no salt and low salt recovered quickly. At milk-ripe stage and under no salt and low salt conditions, the root- and shoot dry masses, leaf area, total root length, total root surface area, root activity, leaf chlorophyll content, and ear leaf net photosynthetic rate, stomatal conductance, transpiration rate, and CAT and POD activities of the cultivars with partial root excision were significantly larger than those of the cultivars with no root excision, while the shoot diameter and ear leaf MDA content were in adverse. Moderate and high salt stresses had greater effects on the cultivars with partial root excision. The root- and shoot dry masses, root morphology, and photosynthesis indices of the cultivars with partial root excision were smaller than those of the cultivars with no root excision, so did the grain yields. Throughout the growth period of the cultivars, the growth of the cultivars with partial root excision depended on the salt concentration, i. e., was promoted under no and low salt, and inhibited under moderate and high salt conditions.

  16. UV protective effects of DNA repair enzymes and RNA lotion.

    PubMed

    Ke, Malcolm S; Camouse, Melissa M; Swain, Freddie R; Oshtory, Shaheen; Matsui, Mary; Mammone, Thomas; Maes, Daniel; Cooper, Kevin D; Stevens, Seth R; Baron, Elma D

    2008-01-01

    Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.

  17. Single Nucleotide Polymorphisms in Nucleotide Excision Repair Genes, Cigarette Smoking, and the Risk of Head and Neck Cancer

    PubMed Central

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

    2013-01-01

    Background Cigarette smoking is associated with increased head and neck cancer (HNC) risk. Tobacco-related carcinogens are known to cause bulky DNA adducts. Nucleotide excision repair (NER) genes encode enzymes that remove adducts and may be independently associated with HNC, as well as modifiers of the association between smoking and HNC. Methods Using population-based case-control data from the Carolina Head and Neck Cancer Epidemiology Study (1,227 cases, 1,325 controls), race-stratified (white, African American) conventional and hierarchical logistic regression models were utilized to estimate odds ratios (OR) with 95% intervals (I) for the independent and joint effects of cigarette smoking and 84 single nucleotide polymorphisms (SNPs) from 15 NER genes on HNC risk. Results The odds of HNC were elevated among ever cigarette smokers, and increased with smoking duration and frequency. Among whites, rs4150403 on ERCC3 was associated with increased HNC odds (AA+AG vs. GG, OR=1.28, 95% I=1.01,1.61). Among African Americans, rs4253132 on ERCC6 was associated with decreased HNC odds (CC+CT vs. TT, OR=0.62, 95% I=0.45,0.86). Interactions between ever cigarette smoking and three SNPs (rs4253132 on ERCC6, rs2291120 on DDB2, and rs744154 on ERCC4) suggested possible departures from additivity among whites. Conclusions We did not find associations between some previously studied NER variants and HNC. We did identify new associations between two SNPs and HNC and three suggestive cigarette-SNP interactions to consider in future studies. Impact We conducted one of the most comprehensive evaluations of NER variants, identifying a few SNPs from biologically plausible candidate genes associated with HNC and possibly interacting with cigarette smoking. PMID:23720401

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

    PubMed Central

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

    2014-01-01

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

  19. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    SciTech Connect

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  20. Association of base excision repair gene polymorphisms with ESRD risk in a Chinese population.

    PubMed

    Cai, Zhenming; Chen, Huimei; Tao, Jing; Guo, Wenwen; Liu, Xiufang; Zheng, Bixia; Sun, Wei; Wang, Yaping

    2012-01-01

    The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/10(6) dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process.

  1. Activation of GLP-1 Receptor Enhances Neuronal Base Excision Repair via PI3K-AKT-Induced Expression of Apurinic/Apyrimidinic Endonuclease 1

    PubMed Central

    Yang, Jenq-Lin; Chen, Wei-Yu; Chen, Yin-Ping; Kuo, Chao-Ying; Chen, Shang-Der

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that increases cellular glucose up-take to decrease blood sugar. Recent studies, however, suggest that the function of GLP-1 is not only to decrease blood sugar, but also acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, and protects synaptic plasticity and memory formation from effects of β-amyloid. Oxidative DNA damage occurs during normal neuron-activity and in many neurological diseases. Our study describes how GLP-1 affected the ability of neurons to ameliorate oxidative DNA damage. We show that activation of GLP-1 receptor (GLP-1R) protect cortical neurons from menadione induced oxidative DNA damage via a signaling pathway involving enhanced DNA repair. GLP-1 stimulates DNA repair by activating the cyclic AMP response element binding protein (CREB) which, consequently, induces the expression of apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision DNA repair (BER) pathway. In this study, APE1 expression was down-regulated as a consequence phosphatidylinositol-3 kinase (PI3K) suppression by the inhibitor LY294002, but not by the suppression of MEK activity. Ischemic stroke is typically caused by overwhelming oxidative-stress in brain cells. Administration of exentin-4, an analogue of GLP-1, efficiently enhanced DNA repair in brain cells of ischemic stroke rats. Our study suggests that a new function of GLP-1 is to elevate DNA repair by inducing the expression of the DNA repair protein APE1. PMID:27698937

  2. Activation of GLP-1 Receptor Enhances Neuronal Base Excision Repair via PI3K-AKT-Induced Expression of Apurinic/Apyrimidinic Endonuclease 1

    PubMed Central

    Yang, Jenq-Lin; Chen, Wei-Yu; Chen, Yin-Ping; Kuo, Chao-Ying; Chen, Shang-Der

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that increases cellular glucose up-take to decrease blood sugar. Recent studies, however, suggest that the function of GLP-1 is not only to decrease blood sugar, but also acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, and protects synaptic plasticity and memory formation from effects of β-amyloid. Oxidative DNA damage occurs during normal neuron-activity and in many neurological diseases. Our study describes how GLP-1 affected the ability of neurons to ameliorate oxidative DNA damage. We show that activation of GLP-1 receptor (GLP-1R) protect cortical neurons from menadione induced oxidative DNA damage via a signaling pathway involving enhanced DNA repair. GLP-1 stimulates DNA repair by activating the cyclic AMP response element binding protein (CREB) which, consequently, induces the expression of apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision DNA repair (BER) pathway. In this study, APE1 expression was down-regulated as a consequence phosphatidylinositol-3 kinase (PI3K) suppression by the inhibitor LY294002, but not by the suppression of MEK activity. Ischemic stroke is typically caused by overwhelming oxidative-stress in brain cells. Administration of exentin-4, an analogue of GLP-1, efficiently enhanced DNA repair in brain cells of ischemic stroke rats. Our study suggests that a new function of GLP-1 is to elevate DNA repair by inducing the expression of the DNA repair protein APE1.

  3. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    PubMed

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  4. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    SciTech Connect

    Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi; Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok; Kang, Tae-Hong

    2015-06-05

    Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.

  5. Alternative excision repair of ultraviolet B- and C-induced DNA damage in dormant and developing spores of Bacillus subtilis.

    PubMed

    Ramírez-Guadiana, Fernando H; Barraza-Salas, Marcelo; Ramírez-Ramírez, Norma; Ortiz-Cortés, Mayte; Setlow, Peter; Pedraza-Reyes, Mario

    2012-11-01

    The nucleotide excision repair (NER) and spore photoproduct lyase DNA repair pathways are major determinants of Bacillus subtilis spore resistance to UV radiation. We report here that a putative ultraviolet (UV) damage endonuclease encoded by ywjD confers protection to developing and dormant spores of B. subtilis against UV DNA damage. In agreement with its predicted function, a His(6)-YwjD recombinant protein catalyzed the specific incision of UV-irradiated DNA in vitro. The maximum expression of a reporter gene fusion to the ywjD opening reading frame occurred late in sporulation, and this maximal expression was dependent on the forespore-specific RNA polymerase sigma factor, σ(G). Although the absence of YwjD and/or UvrA, an essential protein of the NER pathway, sensitized developing spores to UV-C, this effect was lower when these cells were treated with UV-B. In contrast, UV-B but not UV-C radiation dramatically decreased the survival of dormant spores deficient in both YwjD and UvrA. The distinct range of lesions generated by UV-C and UV-B and the different DNA photochemistry in developing and dormant spores may cause these differences. We postulate that in addition to the UvrABC repair system, developing and dormant spores of B. subtilis also rely on an alternative excision repair pathway involving YwjD to deal with the deleterious effects of various UV photoproducts.

  6. Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes

    SciTech Connect

    Hays, J.B.; Ackerman, E.J.; Pang, Q.S. )

    1990-07-01

    Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

  7. Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response.

    PubMed

    Marteijn, Jurgen A; Bekker-Jensen, Simon; Mailand, Niels; Lans, Hannes; Schwertman, Petra; Gourdin, Audrey M; Dantuma, Nico P; Lukas, Jiri; Vermeulen, Wim

    2009-09-21

    Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)-dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV-DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)-DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle-independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia-mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage-induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification. PMID:19797077

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

    PubMed

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

    1997-10-01

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

  9. Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

    PubMed

    Jobert, Laure; Nilsen, Hilde

    2014-07-01

    The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

  10. Case–control analysis of nucleotide excision repair pathway and the risk of renal cell carcinoma

    PubMed Central

    Lin, Jie; Pu, Xia; Wang, Wei; Matin, Surena; Tannir, Nizar M.; Wood, Christopher G.; Wu, Xifeng

    2008-01-01

    In this population-based case–control study with 325 Caucasian renal cell carcinoma (RCC) patients and 335 controls matched to cases by age, gender and county of residence, we evaluated the associations between 13 potential functional polymorphisms in nine major nucleotide excision repair (NER) genes and RCC risk. In individual single nucleotide polymorphism analysis, after adjustment for multiple comparisons, a significantly decreased RCC risk was observed for the heterozygous genotype of XPD Asp312Asn [odds ratio (OR) = 0.62; 95% confidence interval (CI): 0.43–0.90] and for the heterozygous and homozygous variant genotypes combined in a dominant model (OR = 0.64; 95% CI: 0.46–0.89). The heterozygous AG genotype of XPA 5′untranslated region was at 1.78-fold increased risk (95% CI: 1.18–2.69) and the risk reached 2.43-fold (95% CI: 1.57–3.75) for the homozygous variant GG genotype; the risk was significant both in the dominant model and in the recessive model. In joint analysis, compared with individuals with fewer than five adverse alleles, individuals with five (OR = 1.17; 95% CI: 0.71–1.93), six (OR = 1.66; 95% CI: 1.03–2.67), seven or more (OR = 1.85; 95% CI: 1.16–2.95) exhibited a progressively increased risk of RCC (P for trend = 0.004). Further, there were significant interactions between NER pathway genes and sex, hypertension and obesity (all P for interaction <0.05). Our results strongly support that common sequence variants of the NER pathway genes predispose susceptible individuals to increased risk of RCC and that the association may be modified by gender, history of hypertension and obesity. These results need to be replicated in larger studies. PMID:18711149

  11. Trypanosoma brucei harbors a divergent XPB helicase paralog that is specialized in nucleotide excision repair and conserved among kinetoplastid organisms

    PubMed Central

    Badjatia, Nitika; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur

    2013-01-01

    Summary Conserved from yeast to humans, TFIIH is essential for RNA polymerase II transcription and nucleotide excision repair (NER). TFIIH consists of a core that includes the DNA helicase Xeroderma pigmentosum B (XPB) and a kinase subcomplex. Trypanosoma brucei TFIIH harbors all core complex components and is indispensable for RNA polymerase II transcription of spliced leader RNA genes (SLRNAs). Kinetoplastid organisms, however, possess two highly divergent XPB paralogs with only the larger being identified as a TFIIH subunit in T. brucei. Here we show that a knockout of the gene for the smaller paralog, termed XPB-R (R for repair) resulted in viable cultured trypanosomes that grew slower than normal. XPB-R depletion did not affect transcription in vivo or in vitro and XPB-R was not found to occupy the SLRNA promoter which assembles a RNA polymerase II transcription pre-initiation complex including TFIIH. However, XPB-R−/− cells were much less tolerant than wild-type cells to UV light- and cisplatin-induced DNAdamage, which require NER. Since XPB-R−/− cells were not impaired in DNA base excision repair, XPB-R appears to function specifically in NER. Interestingly, several other protists possess highly divergent XPB paralogs suggesting that XPBs specialized in transcription or NER exist beyond the Kinetoplastida. PMID:24134817

  12. Trypanosoma brucei harbours a divergent XPB helicase paralogue that is specialized in nucleotide excision repair and conserved among kinetoplastid organisms.

    PubMed

    Badjatia, Nitika; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur

    2013-12-01

    Conserved from yeast to humans, TFIIH is essential for RNA polymerase II transcription and nucleotide excision repair (NER). TFIIH consists of a core that includes the DNA helicase Xeroderma pigmentosum B (XPB) and a kinase subcomplex. Trypanosoma brucei TFIIH harbours all core complex components and is indispensable for RNA polymerase II transcription of spliced leader RNA genes (SLRNAs). Kinetoplastid organisms, however, possess two highly divergent XPB paralogues with only the larger being identified as a TFIIH subunit in T. brucei. Here we show that a knockout of the gene for the smaller paralogue, termed XPB-R (R for repair) resulted in viable cultured trypanosomes that grew slower than normal. XPB-R depletion did not affect transcription in vivo or in vitro and XPB-R was not found to occupy the SLRNA promoter which assembles a RNA polymerase II transcription pre-initiation complex including TFIIH. However, XPB-R(-/-) cells were much less tolerant than wild-type cells to UV light- and cisplatin-induced DNA damage, which require NER. Since XPB-R(-/-) cells were not impaired in DNA base excision repair, XPB-R appears to function specifically in NER. Interestingly, several other protists possess highly divergent XPB paralogues suggesting that XPBs specialized in transcription or NER exist beyond the Kinetoplastida.

  13. Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine.

    PubMed Central

    Jost, J P

    1993-01-01

    Here I show that nuclear extracts of chicken embryos can promote the active demethylation of DNA. The evidence shows that in hemimethylated DNA (i.e., methylated on one strand only) demethylation of 5mCpG occurs through nucleotide excision repair. The first step of demethylation is the formation of specific nicks 5' from 5-methyldeoxycytidine. Nicks are also observed in vitro on symmetrically methylated CpGs (i.e., methylated on both strands) but they result in breakage of the oligonucleotide with no repair. No specific nicks are observed on the nonmethylated CpG. Nicks are strictly 5mCpG specific and do not occur on 5mCpC, 5mCpT, 5mCpA, or 6mApT. The effect of nonspecific nuclease(s) has been ruled out. The nicking of mCpG takes place in the presence of 20 mM EDTA irrespective of the nature of the sequence surrounding the 5mCpG. No methylcytosine glycosylase activity could be detected. The repair is aphidicolin and N-ethylmaleimide resistant, suggesting a repair action by DNA polymerase beta. In extracts of chicken embryos, the excision repair of mCpG is highest between the 6th and the 12th day of development, whereas it is barely detectable in nuclear extracts from different organs of adults. The possible implications of 5mCpG endonuclease activity in active demethylation of DNA during differentiation is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8506318

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

    SciTech Connect

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

    1983-01-01

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

  15. Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.

    PubMed

    Wang, Li-Juan; Ma, Fei; Tang, Bo; Zhang, Chun-Yang

    2016-08-01

    DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis. PMID:27401302

  16. Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells.

    PubMed

    Fortini, P; Pascucci, B; Parlanti, E; Sobol, R W; Wilson, S H; Dogliotti, E

    1998-03-17

    Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.

  17. Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

    PubMed

    Overmeer, René M; Gourdin, Audrey M; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B; Siegal, Gregg; Fousteri, Maria I; Mullenders, Leon H F; Vermeulen, Wim

    2010-10-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA. PMID:20713449

  18. Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment▿

    PubMed Central

    Overmeer, René M.; Gourdin, Audrey M.; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B.; Siegal, Gregg; Fousteri, Maria I.; Mullenders, Leon H. F.; Vermeulen, Wim

    2010-01-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5′ phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA. PMID:20713449

  19. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

    PubMed Central

    Almeida, Karen H.; Sobol, Robert W.

    2007-01-01

    Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

  20. Phloroglucinol enhances the repair of UVB radiation-induced DNA damage via promotion of the nucleotide excision repair system in vitro and in vivo.

    PubMed

    Piao, Mei Jing; Ahn, Mee Jung; Kang, Kyoung Ah; Kim, Ki Cheon; Cha, Ji Won; Lee, Nam Ho; Hyun, Jin Won

    2015-04-01

    Exposure to solar UVB radiation can lead to the formation of DNA lesions such as cyclobutane pyrimidine dimers (CPDs). Nucleotide excision repair (NER) is critical for the repair of CPDs induced by UV radiation. The purpose of this study was to investigate the ability of phloroglucinol to protect against the formation of UVB-induced CPDs in vitro and in vivo. Exposure to UVB radiation increased the number of CPDs in both HaCaT keratinocytes and mouse skin; however, these increases were reduced by treatment with phloroglucinol. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementation 1 (ERCC1), which are essential components of the NER pathway, were reduced following UVB exposure, although phloroglucinol treatment recovered these levels in both HaCaT keratinocytes and mouse skin. Phloroglucinol also inhibited UVB-induced reductions in binding of the transcription factors specificity protein 1 to the XPC promoter. These results demonstrate that phloroglucinol can protect cells against UVB-induced DNA damage by inducing NER. PMID:25766644

  1. Clinicopathologic Significance of Excision Repair Cross-Complementation 1 Expression in Patients Treated With Breast-Conserving Surgery and Radiation Therapy

    SciTech Connect

    Goyal, Sharad; Yang Qifeng

    2010-03-01

    Purpose: The excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in cancers of the head and neck and the lung. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ERCC1 expression in a cohort of early-stage breast cancer patients treated with breast conservation therapy. Methods and Materials: Paraffin specimens from 504 women with early-stage breast cancer treated with breast conservation therapy were constructed into tissue microarrays. The array was stained for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and ERCC1. This was then correlated with clinicopathologic factors and outcomes data. Results: ERCC-1 expression was evaluable in 366 cases (72%). In this group, 32% and 38% of patients received adjuvant chemotherapy and hormonal therapy, respectively. Increased ERCC-1 expression was found to be correlated with ER positivity (p < 0.005), lower T stage (p < 0.017), nodal negativity (p < 0.013), age >50 (p < 0.006), reduced use of adjuvant chemotherapy (p < 0.02), and increased use of adjuvant hormonal therapy (p < 0.004). ERCC1 expression did not correlate with locoregional recurrence-free survival, distant metastasis-free survival, cause-specific survival, or overall survival. In patients who were both ERCC1-negative and -positive, the use of chemotherapy predicted for worse distant metastasis-free survival (p = 0.05 and p = 0.07, respectively) but not cause-specific survival or overall survival. Conclusions: Although ERCC1 expression did not predict for outcome measures in this dataset, overexpression correlated with favorable prognostic factors such as ER positivity, lower T stage, nodal negativity, and age >50. To our knowledge, this is the first study investigating ERCC1 expression in patients receiving adjuvant radiation

  2. An experimental population study of nucleotide excision repair as a risk factor for UVB-induced melanoma.

    PubMed

    Fernandez, André A; Garcia, Rachel; Paniker, Lakshmi; Trono, David; Mitchell, David L

    2011-01-01

    Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB-inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor-bearing and tumor-free fish were given a challenge UVB dose and (6-4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter-individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter-individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.

  3. An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB-induced Melanoma

    PubMed Central

    Fernandez, André A.; Garcia, Rachel; Paniker, Lakshmi; Trono, David; Mitchell, David L

    2012-01-01

    Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in-situ and in-vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor-bearing and tumor-free fish were given a challenge UVB dose and (6-4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter-individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter-individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility. PMID:21143485

  4. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  5. Mutation of DNA Polymerase β R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice

    PubMed Central

    Pan, Feiyan; Zhao, Jing; Zhou, Ting; Kuang, Zhihui; Dai, Huifang; Wu, Huan; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; Hu, Zhigang; He, Lingfeng; Shen, Binghui; Guo, Zhigang

    2016-01-01

    DNA polymerase β (Pol β), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol β mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol β R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol β functioning in maintaining genomic stability and embryo development. PMID:27358192

  6. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    PubMed

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  7. Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark.

    PubMed

    Chaudhari, Vishalsingh R; Vyawahare, Aniket; Bhattacharjee, Swapan K; Rao, Basuthkar J

    2015-03-01

    Plant cells are known to differentiate their responses to stress depending up on the light conditions. We observed that UVC sensitive phenotype of light grown asynchronous Chlamydomonas reinhardtii culture (Light culture: LC) can be converted to relatively resistant form by transfer to dark condition (Dark culture: DC) before UVC exposure. The absence of photosystem II (PSII) function, by either atrazine treatment in wild type or in D1 (psbA) null mutant, conferred UV protection even in LC. We provide an indirect support for involvement of reactive oxygen species (ROS) signalling by showing higher UV survival on exposures to mild dose of H2O2 or Methyl Viologen. Circadian trained culture also showed a rhythmic variation in UV sensitivity in response to alternating light-dark (12 h:12 h) entrainment, with maximum UV survival at the end of 12 h dark and minimum at the end of 12 h light. This rhythm failed to maintain in "free running" conditions, making it a non-circadian phenotype. Moreover, atrazine strongly inhibited rhythmic UV sensitivity and conferred a constitutively high resistance, without affecting internal circadian rhythm marker expression. Dampening of UV sensitivity rhythm in Thymine-dimer excision repair mutant (cc-888) suggested the involvement of DNA repair in this phenomenon. DNA excision repair (ER) assays in cell-free extracts revealed that dark incubated cells exhibit higher ER compared to those growing in light, underscoring the role of ER in conferring differential UV sensitivity in dark versus light incubation. We suggest that multiple factors such as ROS changes triggered by differences in PSII activity, concomitant with differential ER efficiency collectively contribute to light-dark (12 h: 12 h) rhythmicity in C. reinhardtii UV sensitivity.

  8. XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells

    SciTech Connect

    Wu Qiangen; Beland, Frederick A.; Chang, Ching-Wei; Fang Jialong

    2011-03-01

    Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 {mu}M AZT treatment significantly increased [{sup 3}H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

  9. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    PubMed

    Sousa, Mirta M L; Zub, Kamila Anna; Aas, Per Arne; Hanssen-Bauer, Audun; Demirovic, Aida; Sarno, Antonio; Tian, Erming; Liabakk, Nina B; Slupphaug, Geir

    2013-01-01

    Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers

  10. A UV–Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring

    PubMed Central

    Srivas, Rohith; Costelloe, Thomas; Carvunis, Anne-Ruxandra; Sarkar, Sovan; Malta, Erik; Sun, Su Ming; Pool, Marijke; Licon, Katherine; van Welsem, Tibor; van Leeuwen, Fred; McHugh, Peter J.; van Attikum, Haico; Ideker, Trey

    2014-01-01

    SUMMARY Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions amongst 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks. PMID:24360959

  11. A UV-induced genetic network links the RSC complex to nucleotide excision repair and shows dose-dependent rewiring.

    PubMed

    Srivas, Rohith; Costelloe, Thomas; Carvunis, Anne-Ruxandra; Sarkar, Sovan; Malta, Erik; Sun, Su Ming; Pool, Marijke; Licon, Katherine; van Welsem, Tibor; van Leeuwen, Fred; McHugh, Peter J; van Attikum, Haico; Ideker, Trey

    2013-12-26

    Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

  12. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  13. Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases

    PubMed Central

    Woods, Ryan D.; O'Shea, Valerie L.; Chu, Aurea; Cao, Sheng; Richards, Jody L.; Horvath, Martin P.; David, Sheila S.

    2016-01-01

    MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for ‘retaining’ O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer. PMID:26673696

  14. Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.

    PubMed

    Woods, Ryan D; O'Shea, Valerie L; Chu, Aurea; Cao, Sheng; Richards, Jody L; Horvath, Martin P; David, Sheila S

    2016-01-29

    MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer. PMID:26673696

  15. Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.

    PubMed

    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.

  16. POLYMORPHISMS IN THE DNA BASE EXCISION REPAIR GENES APEX1 AND XRCC1 AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...

  17. The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma

    PubMed Central

    Belanger, KarryAnne K.; Ameredes, Bill T.; Boldogh, Istvan

    2016-01-01

    Asthma is characterized by reversible airway narrowing, shortness of breath, wheezing, coughing, and other symptoms driven by chronic inflammatory processes, commonly triggered by allergens. In 90% of asthmatics, most of these symptoms can also be triggered by intense physical activities and severely exacerbated by environmental factors. This condition is known as exercise-induced asthma (EIA). Current theories explaining EIA pathogenesis involve osmotic and/or thermal alterations in the airways caused by changes in respiratory airflow during exercise. These changes, along with existing airway inflammatory conditions, are associated with increased cellular levels of reactive oxygen species (ROS) affecting important biomolecules including DNA, although the underlying molecular mechanisms have not been completely elucidated. One of the most abundant oxidative DNA lesions is 8-oxoguanine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase 1 (OGG1) during the base excision repair (BER) pathway. Whole-genome expression analyses suggest a cellular response to OGG1-BER, involving genes that may have a role in the pathophysiology of EIA leading to mast cell degranulation, airway hyperresponsiveness, and bronchoconstriction. Accordingly, this review discusses a potential new hypothesis in which OGG1-BER-induced gene expression is associated with EIA symptoms. PMID:27524866

  18. Deficiency in Nucleotide Excision Repair Family Gene Activity, Especially ERCC3, Is Associated with Non-Pigmented Hair Fiber Growth

    PubMed Central

    Yu, Mei; Bell, Robert H.; Ho, Maggie M.; Leung, Gigi; Haegert, Anne; Carr, Nicholas; Shapiro, Jerry; McElwee, Kevin J.

    2012-01-01

    We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation. PMID:22615732

  19. Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining

    PubMed Central

    Smeaton, Michael B.; Miller, Paul S.; Ketner, Gary; Hanakahi, Les A.

    2007-01-01

    The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1–5 × 109 cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host–virus interactions and analysis of mutant cell lines. PMID:18073193

  20. Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease.

    PubMed

    Gredilla, Ricardo; Weissman, Lior; Yang, Jenq-Lin; Bohr, Vilhelm A; Stevnsner, Tinna

    2012-04-01

    Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed.

  1. A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation.

    PubMed

    Heidenreich, Erich; Eisler, Herfried; Lengheimer, Theresia; Dorninger, Petra; Steinboeck, Ferdinand

    2010-01-01

    Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolonged cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary cells. The mutation frequency was decreased in Rad16-deficient cells and further decreased in Rad16/Rad26 double-deficient cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in cell cycle-arrested cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating cells.

  2. Unusual sequence effects on nucleotide excision repair of arylamine lesions: DNA bending/distortion as a primary recognition factor

    PubMed Central

    Jain, Vipin; Hilton, Benjamin; Lin, Bin; Patnaik, Satyakam; Liang, Fengting; Darian, Eva; Zou, Yue; MacKerell, Alexander D.; Cho, Bongsup P.

    2013-01-01

    The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*CN- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of ‘stacked’ (S) and ‘B-type’ (B) conformers, whereas the N-acetylated FAAF also samples a ‘wedge’ (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3′-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli. PMID:23180767

  3. 3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

    PubMed Central

    Schuermann, David; Scheidegger, Simon P.; Weber, Alain R.; Bjørås, Magnar; Leumann, Christian J.; Schär, Primo

    2016-01-01

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases. PMID:26733580

  4. Nucleotide excision repair is reduced in oral epithelial tissues compared with skin.

    PubMed

    Mitchell, David; Paniker, Lakshmi; Godar, Dianne

    2012-01-01

    Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm(TM) , EpiGingival(TM) and EpiOral(TM) . We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared with EpiDerm(TM) . Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment.

  5. Nucleotide excision repair is reduced in oral epithelial tissues compared to skin‡

    PubMed Central

    Mitchell, David; Paniker, Lakshmi; Godar, Dianne

    2012-01-01

    Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm™, EpiGingival™, and EpiOral™ (MatTek Corp.). We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared to EpiDerm™. Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment. PMID:22519509

  6. Topical liposomal DNA-repair enzymes in polymorphic light eruption.

    PubMed

    Hofer, Angelika; Legat, Franz J; Gruber-Wackernagel, Alexandra; Quehenberger, Franz; Wolf, Peter

    2011-07-01

    Polymorphic light eruption (PLE) is a very frequent photodermatosis in Europe whose pathogenesis may involve resistance to UV-induced immune suppression and simultaneous immune reactions against skin photoneoantigens. We performed a randomized, double-blind, placebo-controlled intra-individual half-body trial to investigate the protective effect of an after-sun (AS) lotion containing DNA-repair enzymes (photolyase from Anacystis nidulans and Micrococcus luteus extract with endonuclease activity). Fourteen PLE patients were exposed to suberythemal doses of solar-simulated UV radiation on 4 consecutive days at 4 symmetrically located PLE-prone test fields per patient. The test fields were treated with (i) active AS lotion or (ii) a placebo lotion immediately after each UV exposure, or (iii) an SPF30 sunscreen before UV exposure or left untreated. All test fields were exposed to photoactivating blue light 1 h after each UV exposure. As shown by a newly established specific PLE test score (AA + SI + 0.4P [range, 0-12], where AA is affected area score [range, 0-4], SI is skin infiltration score [range, 0-4], and P is pruritus score on a visual analogue scale [range, 0-10]), PLE symptoms were significantly fewer on test sites treated with active AS lotion than on untreated (P = 0.00049) or placebo-treated test sites (P = 0.024). At 144 h after first UV exposure (the time point of maximal PLE symptoms), the mean test scores for untreated, active AS lotion-treated, and placebo-treated test fields were 4.39, 1.73 (61% reduction; 95% confidence interval (CI), 36% to 85%), and 3.20 (27% reduction; 95% CI, 3% to 51%), respectively. Pretreatment with SPF30 sunscreen completely prevented PLE symptoms in all patients. The present results indicate that DNA damage may trigger PLE and that the application of topical liposomes containing DNA repair enzymes to increase DNA repair may effectively prevent PLE.

  7. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    SciTech Connect

    Smith, P.J.

    1986-03-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

  8. Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP endonuclease activity.

    PubMed

    Fortier, Sean; Yang, Xiaojie; Wang, Yi; Bennett, Richard A O; Strauss, Phyllis R

    2009-06-16

    The base excision repair (BER) pathway recognizes and repairs most nonbulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages, and adults. Using a 45-mer double-stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER. Before 3 days postfertilization (dpf), aphidicolin-sensitive polymerases perform most nucleotide insertion. In fact, eggs and early stage embryos lack DNA polymerase-beta protein. After the eggs have hatched at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase-beta, becomes the primary polymerase. Previously, we showed that when the zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early stage embryos and adults are the absence of DNA polymerase-beta, leading to predominance of replicative polymerases, and the presence of backup Mg(2+)-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs fully when the embryos hatch from the chorionic membrane and encounter normal oxidative stress.

  9. Base excision repair genes and risk of lung cancer among San Francisco Bay Area Latinos and African-Americans

    PubMed Central

    Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Wiencke, John K.

    2009-01-01

    Base excision repair (BER) is the primary DNA damage repair mechanism for repairing small base lesions resulting from oxidation and alkylation damage. This study examines the association between 24 single-nucleotide polymorphisms (SNPs) belonging to five BER genes (XRCC1, APEX1, PARP1, MUTYH and OGG1) and lung cancer among Latinos (113 cases and 299 controls) and African-Americans (255 cases and 280 controls). The goal was to evaluate the differences in genetic contribution to lung cancer risk by ethnic groups. Analyses of individual SNPs and haplotypes were performed using unconditional logistic regressions adjusted for age, sex and genetic ancestry. Four SNPs among Latinos and one SNP among African-Americans were significantly (P < 0.05) associated with either risk of all lung cancer or non-small cell lung cancer (NSCLC). However, only the association between XRCC1 Arg399Gln (rs25487) and NSCLC among Latinos (odds ratio associated with every copy of Gln = 1.52; 95% confidence interval: 1.01–2.28) had a false-positive report probability of <0.5. Arg399Gln is a SNP with some functional evidence and has been shown previously to be an important SNP associated with lung cancer, mostly for Asians. Since the analyses were adjusted for genetic ancestry, the observed association between Arg399Gln and NSCLC among Latinos is unlikely to be confounded by population stratification; however, this result needs to be confirmed by additional studies among the Latino population. This study suggests that there are genetic differences in the association between BER pathway and lung cancer between Latinos and African-Americans. PMID:19029194

  10. Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons.

    PubMed

    Svilar, David; Vens, Conchita; Sobol, Robert W

    2012-01-01

    We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

  11. Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain*

    PubMed Central

    Fowler, Anna-Kate; Hewetson, Aveline; Agrawal, Rajiv G.; Dagda, Marisela; Dagda, Raul; Moaddel, Ruin; Balbo, Silvia; Sanghvi, Mitesh; Chen, Yukun; Hogue, Ryan J.; Bergeson, Susan E.; Henderson, George I.; Kruman, Inna I.

    2012-01-01

    The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain. PMID:23118224

  12. Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    PubMed Central

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-01-01

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

  13. Identification of a conserved 5'-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair.

    PubMed

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-02-29

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5'-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2'-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

  14. Formation of isodialuric acid lesion within DNA oligomers via one-electron oxidation of 5-hydroxyuracil: characterization, stability and excision repair

    PubMed Central

    Simon, Philippe; Gasparutto, Didier; Gambarelli, Serge; Saint-Pierre, Christine; Favier, Alain; Cadet, Jean

    2006-01-01

    5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of •OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in thiswork on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and double-stranded oligonucleotides of various lengths were synthesized and then exposed to the oxidizing action of iridium salts. Isodialuric acid was found to be formed almost quantitatively by a one-electron oxidation mechanism for which relevant information was inferred from a freeze-quenched ESR study. Information on the stability of isodialuric acid thus formed and its conversion products in aqueous solutions was also gained from experiments performed at acidic, neutral and alkali pH’s. Moreover, biochemical features dealing with the substrate specificity of several bacterial and yeast base excision repair enzymes to remove isodialuric acid from site-specifically modified DNA fragments were determined. PMID:16885239

  15. Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme.

    PubMed

    Qi, Yan; Spong, Marie C; Nam, Kwangho; Banerjee, Anirban; Jiralerspong, Sao; Karplus, Martin; Verdine, Gregory L

    2009-12-10

    How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway. PMID:20010681

  16. The Shu complex promotes error-free tolerance of alkylation-induced base excision repair products.

    PubMed

    Godin, Stephen K; Zhang, Zhuying; Herken, Benjamin W; Westmoreland, James W; Lee, Alison G; Mihalevic, Michael J; Yu, Zhongxun; Sobol, Robert W; Resnick, Michael A; Bernstein, Kara A

    2016-09-30

    Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion.

  17. The Shu complex promotes error-free tolerance of alkylation-induced base excision repair products

    PubMed Central

    Godin, Stephen K.; Zhang, Zhuying; Herken, Benjamin W.; Westmoreland, James W.; Lee, Alison G.; Mihalevic, Michael J.; Yu, Zhongxun; Sobol, Robert W.; Resnick, Michael A.; Bernstein, Kara A.

    2016-01-01

    Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion. PMID:27298254

  18. Nucleotide Excision Repair of Chemically Stabilized Analogues of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites

    PubMed Central

    2015-01-01

    Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) J. Org. Chem.79, 5948–5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4′-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (∼20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3′-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes. PMID:25208227

  19. Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

    PubMed

    Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum

    2016-04-19

    DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942

  20. Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

    PubMed Central

    Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum

    2016-01-01

    DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 “opens” up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion “opening” is slow (˜5–10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/“twisting”) of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise “twist-open” mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942

  1. Nucleotide excision repair of chemically stabilized analogues of DNA interstrand cross-links produced from oxidized abasic sites.

    PubMed

    Ghosh, Souradyuti; Greenberg, Marc M

    2014-09-23

    Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4'-oxidized (C4-AP) and C5'-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) J. Org. Chem. 79, 5948-5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4'-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (~20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3'-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes.

  2. Nucleotide Excision Repair Factor XPC Enhances DNA Damage-Induced Apoptosis by Downregulating the Antiapoptotic Short Isoform of Caspase-2

    PubMed Central

    Wang, Qi-En; Han, Chunhua; Zhang, Bo; Sabapathy, Kanaga; Wani, Altaf A.

    2012-01-01

    XPC protein is a critical DNA damage recognition factor in nucleotide excision repair (NER) for which genetic deficiency confers a predisposition to cancer. In this study we demonstrate that XPC has a function that is independent of its canonical function in DNA repair, potentially altering the interpretation of how XPC deficiency leads to heightened cancer susceptibility. XPC enhances apoptosis induced by DNA damage in a p53 nullizygous background, acting downstream of mitochondrial permeabilization and upstream of caspase-9 activation in the DNA damage-induced apoptosis cascade. We found that deficiency in XPC upregulated production of the short isoform of caspase-2 (casp-2S). This upregulation occurred at both protein and mRNA levels through repression of the caspase-2 promoter by XPC protein. Targeted RNAi-mediated downregulation of casp-2S enhanced UV-induced apoptosis as well as activation of caspase-9 and caspase-6 in XPC-deficient cells, but not in XPC-proficient cells. In addition, XPC overexpression in various p53-deficient cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Given that casp-2S functions as an anti-apoptotic protein, our findings suggest that XPC enhances DNA damage-induced apoptosis through inhibition of casp-2S transcription. Together, these findings offer a mechanistic foundation to overcome the resistance of highly prevalent p53-deficient tumors to cell death induced by DNA-damaging therapeutic agents, by targeting strategies that inhibit the expression or function of casp-2S. PMID:22174370

  3. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

    SciTech Connect

    Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha; Massey, Thomas E.

    2014-03-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in

  4. Expression of Excision Repair Cross-Complementation Group 1 as Predictive Marker for Nasopharyngeal Cancer Treated With Concurrent Chemoradiotherapy

    SciTech Connect

    Sun, Jong-Mu; Ahn, Myung-Ju; Park, Min Jae; Lee, Hui-Young; Ahn, Jin Seok; Lee, Seungkoo; Kang, Gu; Han, Joungho; Son, Young-Ik; Baek, Chung-Hwan; Ahn, Yong Chan; Park, Keunchil

    2011-07-01

    Purpose: Cisplatin-based concurrent chemoradiotherapy is the standard treatment of nasopharyngeal cancer. The expression of excision repair cross-complementation group 1 (ERCC1) has been reported to be associated with resistance to platinum-based chemotherapy. We evaluated whether ERCC1 expression could predict the treatment response and survival outcome of patients with locally advanced nasopharyngeal cancer who were treated with cisplatin-based concurrent chemoradiotherapy. Methods and Materials: Immunohistochemistry was used to examine the expression of ERCC1 in nasopharyngeal tumor tissue. Patients were categorized into either a resistant or sensitive group depending on their treatment response outcome. A total of 77 patients were assessed in the present study. Results: The resistant and sensitive groups included 25 and 52 patients, respectively. ERCC1 expression was positive in the tumor tissue for 39 of the 77 patients (51%). Significantly more ERCC1-negative tumors were in the sensitive group than in the resistant group (p = .035). In terms of survival outcome, univariate analysis determined that patients with ERCC1-negative tumors had longer disease-free survival (p = .076) and overall survival (p = .013) than patients with ERCC1-positive tumors. Multivariate analysis determined that negative ERCC expression in tumors was an independent predictor for prolonged overall survival (hazard ratio, 0.14; 95% confidence interval, 0.03-0.71). Conclusion: These results suggest that ERCC1 expression might be a useful predictive marker in patients with locally advanced nasopharyngeal cancer who are under consideration for cisplatin-based concurrent chemoradiotherapy.

  5. A community-based study of nucleotide excision repair polymorphisms in relation to risk of non-melanoma skin cancer

    PubMed Central

    Wheless, Lee; Kistner-Griffin, Emily; Jorgensen, Timothy J.; Ruczinski, Ingo; Berthier-Schaad, Yvette; Kessing, Bailey; Hoffman-Bolton, Judith; Francis, Lesley; Shugart, Yin Yao; Strickland, Paul T.; Kao, W.H. Linda; Alani, Rhoda M.; Smith, Michael W.; Alberg, Anthony J.

    2012-01-01

    Nucleotide excision repair (NER) is responsible for protecting DNA in skin cells against ultraviolet radiation-induced damage. Using a candidate pathway approach, a matched case-control study nested within a prospective, community-based cohort was carried out to test the hypothesis that single nucleotide polymorphisms (SNPs) in NER genes are associated with susceptibility to non-melanoma skin cancer (NMSC). Histologically-confirmed cases of NMSC (n=900) were matched to controls (n=900) on age, gender, and skin type. Associations were measured between NMSC and 221 SNPs in 26 NER genes. Using the additive model, two tightly linked functional SNPs in ERCC6 were significantly associated with increased risk of NMSC: rs2228527 (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.20 – 2.05), and rs2228529 (OR 1.57, 95% CI 1.20 – 2.05). These associations were confined to basal cell carcinoma of the skin (BCC) (rs2228529, OR 1.78, 95% CI 1.30 – 2.44; rs2228527 OR 1.78, 95% CI 1.31 – 2.43). These hypothesis-generating findings suggest functional variants in ERCC6 may be associated with an increased risk of NMSC that may be specific to BCC. PMID:22336945

  6. A mechanism for the exclusion of low-fidelity human Y-family DNA polymerases from base excision repair.

    PubMed

    Haracska, Lajos; Prakash, Louise; Prakash, Satya

    2003-11-15

    The human Y-family DNA polymerases, Poliota, Poleta, and Polkappa, function in promoting replication through DNA lesions. However, because of their low fidelity, any involvement of these polymerases in DNA synthesis during base excision repair (BER) would be highly mutagenic. Mechanisms, therefore, must exist to exclude their participation in BER. Here, we show that although Poliota, Poleta, and Polkappa are all able to form a covalent Schiff base intermediate with the 5'-deoxyribose phosphate (5'-dRP) residue that results from the incision of DNA at an abasic site by an AP endonuclease, they all lack the ability for the subsequent catalytic removal of the 5'-dRP group. Instead, the covalent trapping of these polymerases by the 5'-dRP residue inhibits their DNA synthetic activity during BER. The unprecedented ability of these polymerases for robust Schiff base formation without the release of the 5'-dRP product provides a means of preventing their participation in the DNA synthetic step of BER, thereby avoiding the high incidence of mutagenesis and carcinogenesis that would otherwise occur. PMID:14630940

  7. Heterogeneity of excision repair cross-complementation group 1 gene expression in non-small-cell lung cancer patients

    PubMed Central

    SMIRNOV, SERHEY; PASHKEVICH, ANASTASIYA; LIUNDYSHEVA, VALERIYA; BABENKO, ANDREY; SMOLYAKOVA, RAISA

    2015-01-01

    Excision repair cross-complementation group 1 (ERCC1) gene expression analysis is currently used widely in the molecular diagnosis of cancer. According to numerous studies, ERCC1 gene expression correlates with overall survival and effectiveness of chemotherapy with platinum agents. However, the degree of this correlation differs among various studies, with certain authors reporting a complete lack of such a correlation. These contradictions may be attributed to a number of factors, including the heterogeneity of the tumor tissue. In this study, we attempted to assess the degree of genetic heterogeneity exhibited by tissue samples obtained from non-small-cell lung cancer (NSCLC) through the expression of the ERCC1 gene. This study included 25 samples of tumor tissue from patients with a morphologically confirmed NSCLC diagnosis. A total of three randomized sections of each specimen were used. The ERCC1 gene expression was assessed by quantitative polymerase chain reaction (qPCR) in the TaqMan format. When planning the experiment and analysis of qPCR data, the MIQE guidelines were taken into consideration. We established that the coefficient of variation of the relative level of ERCC1 gene expression in the majority of the samples exceeded 33% (P<0.05), indicating the significant heterogeneity of the sample. We also demonstrated that the degree of heterogeneity of the tumor tissue is largely dependent on disease stage. PMID:25469300

  8. Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH▿

    PubMed Central

    Zotter, Angelika; Luijsterburg, Martijn S.; Warmerdam, Daniël O.; Ibrahim, Shehu; Nigg, Alex; van Cappellen, Wiggert A.; Hoeijmakers, Jan H. J.; van Driel, Roel; Vermeulen, Wim; Houtsmuller, Adriaan B.

    2006-01-01

    The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. PMID:17000769

  9. A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

    PubMed

    Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A; Eichman, Brandt F

    2016-09-14

    DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair. PMID:27571247

  10. Role of the DNA base excision repair protein, APE1 in cisplatin, oxaliplatin, or carboplatin induced sensory neuropathy.

    PubMed

    Kelley, Mark R; Jiang, Yanlin; Guo, Chunlu; Reed, April; Meng, Hongdi; Vasko, Michael R

    2014-01-01

    Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE

  11. Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

    PubMed Central

    Kelley, Mark R.; Jiang, Yanlin; Guo, Chunlu; Reed, April; Meng, Hongdi; Vasko, Michael R.

    2014-01-01

    Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE

  12. A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair

    PubMed Central

    Ranes, Michael; Boeing, Stefan; Wang, Yuming; Wienholz, Franziska; Menoni, Hervé; Walker, Jane; Encheva, Vesela; Chakravarty, Probir; Mari, Pierre-Olivier; Stewart, Aengus; Giglia-Mari, Giuseppina; Snijders, Ambrosius P.; Vermeulen, Wim; Svejstrup, Jesper Q.

    2016-01-01

    Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein. PMID:27060134

  13. The NR4A2 Nuclear Receptor Is Recruited to Novel Nuclear Foci in Response to UV Irradiation and Participates in Nucleotide Excision Repair

    PubMed Central

    Harrison, Matthew; Lim, Wen; Muscat, George E. O.; Sturm, Richard A.; Smith, Aaron G.

    2013-01-01

    Ultraviolet radiation (UVR) is one of the most common mutagens encountered by humans and induces the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproduct (6-4PP) lesions in the genomic DNA. To prevent the accumulation of deleterious mutations these lesions must be efficiently repaired, primarily by nucleotide excision repair. We have previously demonstrated that the NR4A family of nuclear receptors are crucial mediators of the DNA repair function of the MC1R signalling pathway in melanocytes. Here we explore the role of the NR4A2 protein in the DNA repair process further. Using EYFP tagged-NR4A2 we have demonstrated a UVR induced recruitment to distinct nuclear foci where they co-localise with known DNA repair proteins. We reveal that the N-terminal domain of the receptor is required for this translocation and identify a role for p38 and PARP signalling in this process. Moreover disruption of the functional integrity of the Ligand Binding Domain of the receptor by deleting the terminal helix 12 effectively blocks co-localisation of the receptor with DNA repair factors. Restored co-localisation of the mutant receptor with DNA repair proteins in the presence of a Histone Deacetylase Inhibitor suggests that impaired chromatin accessibility underpins the mis-localisation observed. Finally NR4A2 over-expression facilitated a more efficient clearance of UVR induced CPD and 6-4PP lesions. Taken together these data uncover a novel role for the NR4A nuclear receptors as direct facilitators of nucleotide excision repair. PMID:24223135

  14. Metal binding mediated conformational change of XPA protein:a potential cytotoxic mechanism of nickel in the nucleotide excision repair.

    PubMed

    Hu, Jianping; Hu, Ziheng; Zhang, Yan; Gou, Xiaojun; Mu, Ying; Wang, Lirong; Xie, Xiang-Qun

    2016-07-01

    Nucleotide excision repair (NER) is a pivotal life process for repairing DNA nucleotide mismatch caused by chemicals, metal ions, radiation, and other factors. As the initiation step of NER, the xeroderma pigmentosum complementation group A protein (XPA) recognizes damaged DNA molecules, and recruits the replication protein A (RPA), another important player in the NER process. The stability of the Zn(2+)-chelated Zn-finger domain of XPA center core portion (i.e., XPA98-210) is the foundation of its biological functionality, while the displacement of the Zn(2+) by toxic metal ions (such as Ni(2+), a known human carcinogen and allergen) may impair the effectiveness of NER and hence elevate the chance of carcinogenesis. In this study, we first calculated the force field parameters for the bonded model in the metal center of the XPA98-210 system, showing that the calculated results, including charges, bonds, angles etc., are congruent with previously reported results measured by spectrometry experiments and quantum chemistry computation. Then, comparative molecular dynamics simulations using these parameters revealed the changes in the conformation and motion mode of XPA98-210 Zn-finger after the substitution of Zn(2+) by Ni(2+). The results showed that Ni(2+) dramatically disrupted the relative positions of the four Cys residues in the Zn-finger structure, forcing them to collapse from a tetrahedron into an almost planar structure. Finally, we acquired the binding mode of XPA98-210 with its ligands RPA70N and DNA based on molecular docking and structural alignment. We found that XPA98-210's Zn-finger domain primarily binds to a V-shaped cleft in RPA70N, while the cationic band in its C-terminal subdomain participates in the recognition of damaged DNA. In addition, this article sheds light on the multi-component interaction pattern among XPA, DNA, and other NER-related proteins (i.e., RPA70N, RPA70A, RPA70B, RPA70C, RPA32, and RPA14) based on previously reported

  15. The role of base excision repair in the development of primary open angle glaucoma in the Polish population.

    PubMed

    Cuchra, Magda; Markiewicz, Lukasz; Mucha, Bartosz; Pytel, Dariusz; Szymanek, Katarzyna; Szemraj, Janusz; Szaflik, Jerzy; Szaflik, Jacek P; Majsterek, Ireneusz

    2015-08-01

    Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399 Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02-1.89 p = 0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399 Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07-2.61 P = 0.02) clinical parameter. Moreover, the association of VF factor with 148 Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30-3.89) was also found. Additionally, the analysis of the 324 Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might decrease the progression of POAG (OR 0.47; 95% CI 0.30-0.82 P = 0.005). We suggest that the 399 Arg/Gln polymorphism of the XRCC1 gene may serve as a predictive risk factor of POAG.

  16. Nucleotide excision repair genes and risk of lung cancer among San Francisco bay area Latinos and African Americans

    PubMed Central

    Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Kittles, Rick A.; Silva, Gabriel; Wiencke, John K.

    2009-01-01

    Few studies on the association between nucleotide excision repair (NER) variants and lung cancer risk have included Latinos and African Americans. We examine variants in six NER genes (ERCC2, ERCC4, ERCC5, LIG1, RAD23B and XPC) in association with primary lung cancer risk among 113 Latino and 255 African American subjects newly diagnosed with primary lung cancer from 1998 to 2003 in the San Francisco Bay Area, and 579 healthy controls (299 Latinos and 280 African Americans). Individual single nucleotide polymorphism and haplotype analyses, multifactor dimensionality reduction, and principal components analysis were performed to assess the association between six genes in the NER pathway and lung cancer risk. Among Latinos, ERCC2 haplotype CGA (rs238406, rs11878644, rs6966) was associated with reduced lung cancer risk [odds ratio (OR) of 0.65 and 95% confidence interval (CI): 0.44-0.97], especially among non-smokers (OR=0.29; 95% CI: 0.12-0.67). From multifactor dimensionality reduction analysis, in Latinos, smoking and three SNPs (ERCC2 rs171140, ERCC5 rs17655, and LIG1 rs20581) together had a prediction accuracy of 67.4% (p=0.001) for lung cancer. Among African Americans, His/His genotype of ERCC5 His1104Asp (rs17655) was associated with increased lung cancer risk (OR=1.78; 95% CI: 1.09-2.91), and LIG1 haplotype GGGAA (rs20581, rs156641, rs3730931, rs20579, and rs439132) was associated with reduced lung cancer risk (OR=0.61; 95% CI: 0.42-0.88). Our study suggests different elements of the NER pathway may be important in the different ethnic groups resulting either from different linkage relationship, genetic backgrounds, and/or exposure histories. PMID:18709642

  17. Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

    PubMed

    Jordheim, Lars Petter; Cros-Perrial, Emeline; Matera, Eva-Laure; Bouledrak, Karima; Dumontet, Charles

    2014-10-01

    Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

  18. Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

    PubMed Central

    Maciejewski, Sonia; Nguyen, Joseph H. C.; Gómez-Herreros, Fernando; Cortés-Ledesma, Felipe; Caldecott, Keith W.

    2015-01-01

    ABSTRACT Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. PMID:26715620

  19. Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies.

    PubMed

    Yarosh, D B; O'Connor, A; Alas, L; Potten, C; Wolf, P

    1999-02-01

    A new approach to photoprotection is to repair DNA damage after UV exposure. This can be accomplished by delivery of a DNA repair enzyme with specificity to UV-induced cyclobutane pyrimidine dimers into skin by means of specially engineered liposomes. Treatment of DNA-repair-deficient xeroderma pigmentosum patients or skin cancer patients with T4N5 liposome lotion containing such DNA repair liposomes increases the removal of DNA damage in the first few hours after treatment. In these studies, a DNA repair effect was observed in some patients treated with heat-inactivated enzyme. Unexpectedly, it was discovered that the heat-inactivated T4 endonuclease V enzyme refolds and recovers enzymatic activity. These studies demonstrate that measurements of molecular changes induced by biological drugs are useful adjuvants to clinical studies.

  20. Prospective randomized controlled study of excision versus distal splitting of hernial sac and processus vaginalis in the repair of inguinal hernias and communicating hydroceles.

    PubMed

    Gahukamble, D B; Khamage, A S

    1995-04-01

    A total of 798 pediatric patients between the ages of 16 days and 10 years underwent a randomized trial of two surgical procedures to assess the superiority of one over the other. In the first group of 231 patients, 236 hernial sacs (HS)/processus vaginalis (PV) were excised completely after transfixation and transection of the sac at the internal ring, whereas in the second group of 567 patients, 595 residual HS/PV were not excised but split longitudinally. The results showed that there was no significant difference in the two groups as far as complications were concerned. Considering the results of these two procedures, it could be concluded that even the less extensive process of distal longitudinal splitting of the residual HS or PV can be preferred in the repair of hernias or communicating hydroceles in male children.

  1. Recognition of Damaged DNA for Nucleotide Excision Repair: A Correlated Motion Mechanism with a Mismatched cis-syn Thymine Dimer Lesion

    PubMed Central

    2015-01-01

    Mammalian global genomic nucleotide excision repair requires lesion recognition by XPC, whose detailed binding mechanism remains to be elucidated. Here we have delineated the dynamic molecular pathway and energetics of lesion-specific and productive binding by the Rad4/yeast XPC lesion recognition factor, as it forms the open complex [Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570–575; Chen, X., et al. (2015) Nat. Commun. 6, 5849] that is required for excision. We investigated extensively a cis-syn cyclobutane pyrimidine dimer in mismatched duplex DNA, using high-level computational approaches. Our results delineate a preferred correlated motion mechanism, which provides for the first time an atomistic description of the sequence of events as Rad4 productively binds to the damaged DNA. PMID:26270861

  2. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

    PubMed Central

    Carr, A M; Sheldrick, K S; Murray, J M; al-Harithy, R; Watts, F Z; Lehmann, A R

    1993-01-01

    Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2. Images PMID:8464724

  3. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes.

    PubMed

    Piao, Mei Jing; Hewage, Susara Ruwan Kumara Madduma; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Lee, Nam Ho; Hyun, Jin Won

    2015-09-02

    We investigated the protective properties of diphlorethohydroxycarmalol (DPHC), a phlorotannin, against ultraviolet B (UVB) radiation-induced cyclobutane pyrimidine dimers (CPDs) in HaCaT human keratinocytes. The nucleotide excision repair (NER) system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementing 1 (ERCC1), which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1) and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

  4. Serum levels of renin, angiotensin-converting enzyme and angiotensin II in patients treated by surgical excision, propranolol and captopril for problematic proliferating infantile haemangioma.

    PubMed

    Sulzberger, L; Baillie, R; Itinteang, T; de Jong, S; Marsh, R; Leadbitter, P; Tan, S T

    2016-03-01

    The role of the renin-angiotensin system (RAS) in the biology of infantile haemangioma (IH) and its accelerated involution induced by β-blockers was first proposed in 2010. This led to the first clinical trial in 2012 using low-dose captopril, an angiotensin-converting enzyme (ACE) inhibitor, demonstrating a similar response in these tumours. This study aimed to compare serial serum levels of the components of the RAS in patients before and after surgical excision, propranolol or captopril treatment for problematic proliferating IH. Patients with problematic proliferating IH underwent measurements of serum levels of plasma renin activity (PRA), ACE and angiotensin II (ATII) before, and 1-2 and 6 months following surgical excision, propranolol or captopril treatment. This study included 27 patients undergoing surgical excision (n = 8), propranolol (n = 11) and captopril (n = 8) treatment. Treatment with either surgical excision or propranolol resulted in significant decrease in the mean levels of PRA. Surgical excision or captopril treatment led to significant decline in the mean levels of ATII. All three treatment modalities had no significant effect on the mean levels of ACE. This study demonstrates the effect of surgical excision, propranolol and captopril treatment in lowering the levels of PRA and ATII, but not ACE, supporting a mechanistic role for the RAS in the biology of IH.

  5. Serum levels of renin, angiotensin-converting enzyme and angiotensin II in patients treated by surgical excision, propranolol and captopril for problematic proliferating infantile haemangioma.

    PubMed

    Sulzberger, L; Baillie, R; Itinteang, T; de Jong, S; Marsh, R; Leadbitter, P; Tan, S T

    2016-03-01

    The role of the renin-angiotensin system (RAS) in the biology of infantile haemangioma (IH) and its accelerated involution induced by β-blockers was first proposed in 2010. This led to the first clinical trial in 2012 using low-dose captopril, an angiotensin-converting enzyme (ACE) inhibitor, demonstrating a similar response in these tumours. This study aimed to compare serial serum levels of the components of the RAS in patients before and after surgical excision, propranolol or captopril treatment for problematic proliferating IH. Patients with problematic proliferating IH underwent measurements of serum levels of plasma renin activity (PRA), ACE and angiotensin II (ATII) before, and 1-2 and 6 months following surgical excision, propranolol or captopril treatment. This study included 27 patients undergoing surgical excision (n = 8), propranolol (n = 11) and captopril (n = 8) treatment. Treatment with either surgical excision or propranolol resulted in significant decrease in the mean levels of PRA. Surgical excision or captopril treatment led to significant decline in the mean levels of ATII. All three treatment modalities had no significant effect on the mean levels of ACE. This study demonstrates the effect of surgical excision, propranolol and captopril treatment in lowering the levels of PRA and ATII, but not ACE, supporting a mechanistic role for the RAS in the biology of IH. PMID:26612192

  6. Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

    PubMed Central

    Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia

    2014-01-01

    Abstract Significance: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. Recent Advances: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. Critical Issues: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. Future Directions: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world. Antioxid. Redox Signal. 20, 621–639. PMID:23879289

  7. Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A*

    PubMed Central

    Shell, Steven M.; Li, Zhengke; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka; Brosey, Chris; Serrano, Moises A.; Chazin, Walter J.; Musich, Phillip R.; Zou, Yue

    2009-01-01

    In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation. PMID:19586908

  8. The Saccharomyces cerevisiae RAD7 and RAD16 genes are required for inducible excision of endonuclease III sensitive-sites, yet are not needed for the repair of these lesions following a single UV dose.

    PubMed

    Scott, A D; Waters, R

    1997-01-31

    The RAD7 and RAD16 genes of Saccharomyces cerevisiae have roles in the repair of UV induced CPDs in nontranscribed genes [1], and in the repair of CPDs in the nontranscribed strand of transcribed genes [2]. Previously, we identified an inducible component to nucleotide excision repair (NER), which is absent in a rad16 delta strain [3]. We have examined the repair of UV induced endonuclease III sensitive-sites (EIIISS), and have shown repair of these lesions to proceed by NER but their removal from nontranscribed regions is independent of RAD7 and RAD16. Furthermore, EIIISS are repaired with equal efficiency from both transcribed and nontranscribed genes [4]. In order to dissect the roles of RAD7 and RAD16 in the above processes we examined the repair of EIIISS in the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. These loci have elevated levels of these lesions after UV (in genomic DNA EIIISS constitute about 10% of total lesions, whereas CPDs are about 70% of total lesions). We have shown that excision of UV induced EIIISS is enhanced following a prior UV irradiation. No enhancement of repair was detected in either the rad7 delta or the rad16 delta mutant. The fact that RAD7 and RAD16 are not required for the repair of EIIISS per se yet are required for the enhanced excision of these lesions from MAT alpha and HML alpha suggests two possibilities. These genes have two roles in NER, namely in the repair of CPDs from nontranscribed sequences, and in enhancing NER itself regardless of whether these genes' products are required for the excision of the specific lesion being repaired. In the latter case, the induction of RAD7 and RAD16 may increase the turnover of complexes stalled in nontranscribed DNA so as to increase the availability of NER proteins for the repair of CPDs and EIIISS in all regions of the genome.

  9. Comparative study of the application of microcurrent and AsGa 904 nm laser radiation in the process of repair after calvaria bone excision in rats

    NASA Astrophysics Data System (ADS)

    Mendonça, J. S.; Neves, L. M. G.; Esquisatto, M. A. M.; Mendonça, F. A. S.; Santos, G. M. T.

    2013-03-01

    This study evaluated the effects of microcurrent stimulation (10 μA/5 min) and 904 nm GaAs laser irradiation (3 J cm-2 for 69 s/day) on excisional lesions created in the calvaria bone of Wistar rats. The results showed significant responses in the reduction of inflammatory cells and an increase in the number of new blood vessels, number of fibroblasts and deposition of birefringent collagen fibers when these data were compared with those of samples of the untreated lesions. Both applications, microcurrent and laser at 904 nm, favored tissue repair in the region of bone excisions during the study period and these techniques can be used as coadjuvantes in the repair of bone tissue.

  10. Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts.

    PubMed

    Roza, L; Vermeulen, W; Bergen Henegouwen, J B; Eker, A P; Jaspers, N G; Lohman, P H; Hoeijmakers, J H

    1990-03-15

    UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, e.g., (6-4) photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

  11. Multimodality gynecomastia repair by cross-chest power-assisted superficial liposuction combined with endoscopic-assisted pull-through excision.

    PubMed

    Ramon, Ytzhack; Fodor, Lucian; Peled, Isaac J; Eldor, Liron; Egozi, Dana; Ullmann, Yehuda

    2005-12-01

    Numerous methods of gynecomastia repair have been described to accomplish removal of breast tissue. Our multimodality surgical approach for the treatment of gynecomastia combines the use of power-assisted superficial cross-chest liposuction with direct pull-through excision of the breast parenchyma under endoscopic supervision. Seventeen patients, aging 17-39, underwent this multimodality approach. According to Simon's grading, 3 patients had grade 1, 5 had grade 2a, 6 had grade 2b, and 3 had grade 3 gynecomastia. Power-assisted liposuction was performed with a 3- or 4-mm triple-hole cannula inserted through the contralateral periareolar medial incision to suction the contralateral prepectoral fatty breast. At the end of the liposuction, the fibrous tissue was easily pulled through the ipsilateral stab wound and excised under endoscopic control. Follow-up time ranged from 6 to 34 months. The amount of fat removed by liposuction varied from 100-800 mL per breast, and the amount of breast parenchyma removed by excision varied from 20-110 g. All patients recovered remarkably well. No complications were recorded. All patients were satisfied with their results. This technique enables an effective treatment of both the fatty and fibrous tissue of the male breast and avoids skin redundancy due to skin contraction. A smooth masculine breast contour is consistently achieved without the stigma of this type of surgery.

  12. Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair.

    PubMed

    Higa, Mitsuru; Zhang, Xue; Tanaka, Kiyoji; Saijo, Masafumi

    2016-06-24

    UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER. PMID:27129218

  13. The Effect of Msh2 Knockdown on Toxicity Induced by tert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

    PubMed Central

    Cooley, N.; Elder, R. H.; Povey, A. C.

    2013-01-01

    The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg+/+, Nth1+/+) and deficient (Mpg−/−, Nth1−/−) mouse embryonic fibroblasts (MEFs) following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1+/+ cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1−/− cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg+/+ cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg−/− cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity. PMID:23984319

  14. Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions.

    PubMed

    Reynolds, Pamela; Cooper, Sarah; Lomax, Martine; O'Neill, Peter

    2015-04-30

    The repair of endogenously induced DNA damage is essential to maintain genomic integrity. It has been shown that XRCC1 and PARP1 are involved in the repair of base lesions and SSBs, although the exact mode of action has yet to be determined. Here we show that XRCC1 is involved in the repair of base lesions and SSBs independent of the cell cycle. However, the rate of repair of damage requiring XRCC1 does reflect the damage complexity. The repair of induced DNA damage occurs by PARP1-dependent and PARP1-independent sub-pathways of BER. It is suggested that the repair of SSBs and purine base damage is by a sub-pathway of BER that requires both XRCC1 and PARP1. Repair of pyrimidine base damage may require XRCC1 but does not require PARP1 activity. Therefore, although BER of simple lesions occurs rapidly, pathway choice and the involvement of PARP1 are highly dependent on the types of lesion induced.

  15. A novel role for Gadd45α in base excision repair: Modulation of APE1 activity by the direct interaction of Gadd45α with PCNA

    SciTech Connect

    Kim, Hye Lim; Kim, Sang Uk; Seo, Young Rok

    2013-05-03

    Highlights: ► Emerging critical role for Gadd45α in modulating BER activity. ► Identifying specific PCNA binding site on Gadd45α protein. ► Regulating APE1 activity through interaction between Gadd45α and PCNA. ► Suggesting potential role of Gadd45α–PCNA binding in pancreatic carcinogenesis. -- Abstract: The growth arrest and DNA damage inducible, alpha (Gadd45α) protein regulates DNA repair by interacting with proliferating cell nuclear antigen (PCNA). Our previous study suggested a potential role for Gadd45α in the base excision repair (BER) pathway by affecting apurinic/apyrimidinic endonuclease 1 (APE1) protein in addition to its accepted role in nucleotide excision repair (NER). Here, we investigated whether the interaction of Gadd45α with PCNA affects APE1 activity. To address this issue, we used a siRNA directed to Gadd45α and a form of Gadd45α with a mutation to the predicted site of PCNA binding. There was a reduction of APE1 activity in cells transfected with the Gadd45α siRNA. Furthermore, the interaction of Gadd45α with PCNA and APE1 was lower in cells transfected with mutant Gadd45α compared with cells transfected with wild-type Gadd45α. Indeed, we observed that the APE1 activity in the Gadd45α-interacting complex was significantly lower in cells that overexpress mutant Gadd45α compared with cells that overexpress wild-type Gadd45α. We conclude that the PCNA binding site on Gadd45α plays a critical role in modulating the interaction with PCNA and APE1, affecting BER activity. These results provide novel insights into the mechanisms by which BER activity is modulated, although the interaction of Gadd45α with APE1 needs to be clarified.

  16. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra.

    PubMed

    Vreeswijk, Maaike P G; Meijers, Caro M; Giphart-Gassler, Micheline; Vrieling, Harry; van Zeeland, Albert A; Mullenders, Leon H F; Loenen, Wil A M

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4 PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C>T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  17. Reduction of the DNA base excision repair protein, XRCC1, may contribute to DNA fragmentation after cold injury-induced brain trauma in mice.

    PubMed

    Fujimura, M; Morita-Fujimura, Y; Noshita, N; Yoshimoto, T; Chan, P H

    2000-06-30

    The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.

  18. Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

    PubMed Central

    He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.

    2014-01-01

    Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis. PMID:25118285

  19. Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

    PubMed

    Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

  20. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle

    PubMed Central

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A.

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation. PMID:27442013

  1. Genetic variations in base excision repair pathway and risk of bladder cancer: a case-control study in the United States.

    PubMed

    Xie, Hui; Gong, Yilei; Dai, Jingyao; Wu, Xifeng; Gu, Jian

    2015-01-01

    Base excision repair (BER) is one of the major cellular DNA repair pathways that repairs small isolated foci of DNA damage including reduced or oxidized single bases or fragments and small, non-bulky adducts. Genetic variations in BER genes may affect DNA repair capacity and increase susceptibility to bladder cancer. In a case-control study of 801 bladder cancer patients and 801 matched controls, we evaluated the associations of 167 single nucleotide polymorphisms (SNPs) from 19 genes of the BER pathway with the risk of bladder cancer. In individual SNP analysis, 13 SNPs in 10 BER pathway genes were significantly associated with bladder cancer risk. The most significant SNP was rs2029167 in the SMUG1 gene. The homozygous variant GG genotype was associated with a 1.42-fold increased risk of bladder cancer (95% confidence interval [CI], 1.11-1.82, P=0.005). Cumulative effect analysis showed joint effects of increased risk of bladder cancer with increasing number of unfavorable genotypes in patients. Classification and regression tree analysis further revealed high-order gene-gene interactions and categorized the study subjects into low-, medium-low-, medium-high-, and high-risk groups. Compared with the low-risk group, the odds ratio for medium-low-, medium-high-, and high-risk group was 1.83 (95% CI: 1.23-2.72), 2.61 (95% CI: 1.79-3.80), and 3.05 (95% CI: 2.08-4.46), respectively (P for trend<0.001). Our results suggest that genetic variations in BER pathway genes modulate the risk of bladder cancer individually and jointly.

  2. UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

    PubMed

    Tanaka, K; Kamiuchi, S; Ren, Y; Yonemasu, R; Ichikawa, M; Murai, H; Yoshino, M; Takeuchi, S; Saijo, M; Nakatsu, Y; Miyauchi-Hashimoto, H; Horio, T

    2001-06-01

    Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP. PMID:11376684

  3. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

    PubMed

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation. PMID:27442013

  4. Naturally occurring polyphenol, morin hydrate, inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme with various roles in human disease.

    PubMed

    Dixon, Monica; Woodrick, Jordan; Gupta, Suhani; Karmahapatra, Soumendra Krishna; Devito, Stephen; Vasudevan, Sona; Dakshanamurthy, Sivanesan; Adhikari, Sanjay; Yenugonda, Venkata M; Roy, Rabindra

    2015-03-01

    Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50=2.6μM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavonoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

  5. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    PubMed

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER.

  6. ABT-888 and quinacrine induced apoptosis in metastatic breast cancer stem cells by inhibiting base excision repair via adenomatous polyposis coli.

    PubMed

    Siddharth, Sumit; Nayak, Deepika; Nayak, Anmada; Das, Sarita; Kundu, Chanakya Nath

    2016-09-01

    PARP inhibitors in combination with other agents are in clinical trial against cancer, but its effect on cancer stem cells (CSCs) is limited. CSCs are responsible for drug resistance, metastasis and cancer relapse due to high DNA repair capacity. Here, we present preclinical effects of Quinacrine (QC) with ABT-888, a PARP inhibitor, on highly metastatic breast cancer stem cells (mBCSCs). An increased level of Adenomatous polyposis coli (APC) was noted after treatment with ABT-888 in QC pre-treated mBCSCs cells. Increased APC physically interacts with PARP-1 and inhibits PARylation causing the non assembly of base excision repair (BER) multiprotein complex, resulting in an irreparable DNA damage and subsequent apoptosis. Knockdown of APC in mBCSCs inhibited DNA damage, increased BER and PARylation, reduces apoptosis while the over-expression of APC in BT20 (APC low expressing) cells reversed the effect. Thus, combination of QC and ABT-888 decreased mBCSCs growth by activating APC and inhibiting BER within the cells. PMID:27334689

  7. Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

    PubMed

    Maira, N; Torres, T M; de Oliveira, A L; de Medeiros, S R B; Agnez-Lima, L F; Lima, J P M S; Scortecci, K C

    2014-05-01

    Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

  8. Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair.

    PubMed

    Gourdin, Audrey M; van Cuijk, Loes; Tresini, Maria; Luijsterburg, Martijn S; Nigg, Alex L; Giglia-Mari, Guiseppina; Houtsmuller, Adriaan B; Vermeulen, Wim; Marteijn, Jurgen A

    2014-12-01

    The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER. PMID:25453469

  9. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    PubMed

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. PMID:26805762

  10. The structure of the human ERCC1/XPF interaction domains reveals a complementary role for the two proteins in nucleotide excision repair.

    PubMed

    Tripsianes, Konstantinos; Folkers, Gert; Ab, Eiso; Das, Devashish; Odijk, Hanny; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf

    2005-12-01

    The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair. PMID:16338413

  11. Cockayne Syndrome: Varied Requirement of Transcription-Coupled Nucleotide Excision Repair for the Removal of Three Structurally Different Adducts from Transcribed DNA

    PubMed Central

    Kitsera, Nataliya; Gasteiger, Karola; Lühnsdorf, Bork; Allgayer, Julia; Epe, Bernd; Carell, Thomas; Khobta, Andriy

    2014-01-01

    Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER) pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS), however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene adduct (dG(N2)-AAF) constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is removed exclusively by the CSA- and CSB-dependent pathway. In contrast, contribution of the CS proteins to the removal of two other transcription-blocking DNA lesions – N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG(C8)-AAF) and cyclobutane thymine-thymine (TT) dimer – is only minor (TT dimer) or none (dG(C8)-AAF). The unique properties of dG(N2)-AAF identify this adduct as a prototype for a new class of DNA lesions that escape the alternative global genome repair and could be critical for the CS pathogenesis. PMID:24713864

  12. Excision without excision

    SciTech Connect

    Brown, David; Sarbach, Olivier; Schnetter, Erik; Diener, Peter; Tiglio, Manuel; Hawke, Ian; Pollney, Denis

    2007-10-15

    to turducken (turduckens, turduckening, turduckened, turduckened) [math.]: To stuff a black hole. We analyze and apply an alternative to black hole excision based on smoothing the interior of black holes with arbitrary initial data, and solving the vacuum Einstein evolution equations everywhere. By deriving the constraint propagation system for our hyperbolic formulation of the BSSN evolution system we rigorously prove that the constraints propagate causally and so any constraint violations introduced inside the black holes cannot affect the exterior spacetime. We present evolutions of Cook-Pfeiffer binary black hole initial configurations showing that these techniques appear to work robustly for generic data. We also present evidence from spherically symmetric evolutions that for the gauge conditions used the same stationary end-state is approached irrespective of the choice of initial data and smoothing procedure.

  13. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    SciTech Connect

    Carles, Joan . E-mail: jcarles@imas.imim.es; Monzo, Mariano; Amat, Marta; Jansa, Sonia; Artells, Rosa; Navarro, Alfons; Foro, Palmira; Alameda, Francesc; Gayete, Angel; Gel, Bernat; Miguel, Maribel; Albanell, Joan; Fabregat, Xavier

    2006-11-15

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC.

  14. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype

    PubMed Central

    Etemadi, Arash; Islami, Farhad; Phillips, David H.; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T; Taylor, Philip R; Boffetta, Paolo; Abnet, Christian C; Dawsey, Sanford M; Malekzadeh, Reza; van Schooten, Frederik J.

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly-selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in 8 DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by 32P-postlabelling. Multivariable regression models were compared by Akaike’s information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 108 nucleotides (mean: 5.8±3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non risk-allele genotype, and was higher in the MPO homozygote risk-allele genotype. The sum of risk alleles in these genes significantly correlated with the log-adduct level (r=0.4, p<0.001). Compared with the environmental model, adding phase I SNPs and NER capacity provided the best fit, and could explain 17% more of the variation in adduct levels. NER capacity was affected by polymorphisms in the MTHFR and ERCC1 genes. Female non-smokers in this population had PAH-related DNA adduct levels 3-4 times higher than smokers and occupationally-exposed groups in previous studies, with large inter-individual variation which could best be explained by a combination of phase I genes and NER capacity. PMID:23175176

  15. UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells.

    PubMed

    Oh, Kyu-Seon; Bustin, Michael; Mazur, Sharlyn J; Appella, Ettore; Kraemer, Kenneth H

    2011-01-01

    DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.

  16. DNA repair enzymes: an important role in skin cancer prevention and reversal of photodamage--a review of the literature.

    PubMed

    Kabir, Yasmeen; Seidel, Rachel; Mcknight, Braden; Moy, Ronald

    2015-03-01

    The incidence of skin cancer continues to increase annually despite preventative measures. Non-melanoma skin cancer affects more than 1,000,000 people in the United States every year.1 The current preventative measures, such as sunscreens and topical antioxidants, have not shown to be effective in blocking the effects of UV radiation based on these statistics. The level of antioxidants contained in the majority of skin creams is not sufficient to majorly impact free radical damage. Sunscreens absorb only a portion of UV radiation and often are not photostable. In this review article, we present the novel use of exogenous DNA repair enzymes and describe their role in combating photocarcinogenesis and photoaging. Topical application of these enzymes serves to supplement intrinsic DNA repair mechanisms. The direct repair of DNA damage by endogenous repair enzymes lessens rates of mutagenesis and strengthens the immune response to tumor cells. However, these innate mechanisms are not 100% efficient. The use of exogenous DNA repair enzymes presents a novel way to supplement intrinsic mechanisms and improve their efficacy. Several DNA repair enzymes critical to the prevention of cutaneous malignancies have been isolated and added to topical preparations designed for skin cancer prevention. These DNA repair enzymes maximize the rate of DNA repair and provide a more efficient response to carcinogenesis.

  17. A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells.

    PubMed

    Rouget, Raphael; Auclair, Yannick; Loignon, Martin; Affar, El Bachir; Drobetsky, Elliot A

    2008-02-29

    In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  20. Crosstalk between MSH2-MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair.

    PubMed

    Lai, Yanhao; Budworth, Helen; Beaver, Jill M; Chan, Nelson L S; Zhang, Zunzhen; McMurray, Cynthia T; Liu, Yuan

    2016-01-01

    Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion. PMID:27546332

  1. The COOH terminus of suppressor of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair.

    PubMed

    Sweder, K S; Hanawalt, P C

    1994-01-21

    We examined several yeast strains with different mutations in the essential SSL2 (Suppressor of Stem Loop, also called RAD25) gene for their ability to remove cyclobutane pyrimidine dimers from expressed genes, and from the genome overall. The SSL2 protein has a high degree of amino acid sequence identity to the protein encoded by the human ERCC3 gene (Gulyas, K. D., and Donahue, T. F. (1992) Cell 69, 1031-0142). The mutant allele SSL2-XP encodes a protein resembling the mutated ERCC3 protein from UV-sensitive human cells belonging to xeroderma pigmentosum complementation group B and Cockayne's syndrome (CS) complementation group C (Weeda, G., van Ham, R. C. A., Vermeulen, W., Bootsma, D., van der Eb, A. J., and Hoeijmakers, J. H. J. (1990) Cell 62, 777-791; Gulyas and Donahue, 1992). The SSL2-XP allele confers UV sensitivity on yeast strain KG119. We found that the biochemical basis for the UV sensitivity of KG119 is a complete deficiency in the removal of cyclobutane pyrimidine dimers from the overall genome as well as a deficiency in transcription-coupled repair. This is the first analysis of the DNA repair defect responsible for the UV sensitivity of cells carrying the SSL2-XP allele, and it documents the similarity of the defect to that associated with XP-B/CS-C, and the difference between this defect and that in cells belonging to CS complementation groups A and B. PMID:8294433

  2. p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

    PubMed

    Lloyd, Daniel R; Hanawalt, Philip C

    2002-09-15

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the

  3. Radio-adaptive response of base excision repair genes and proteins in human peripheral blood mononuclear cells exposed to gamma radiation.

    PubMed

    Toprani, Sneh M; Das, Birajalaxmi

    2015-09-01

    Radio-adaptive response is a mechanism whereby a low-dose exposure (priming dose) induces resistance to a higher dose (challenging dose) thus significantly reducing its detrimental effects. Radiation-induced DNA damage gets repaired through various DNA repair pathways in human cells depending upon the type of lesion. The base excision repair (BER) pathway repairs radiation-induced base damage, abasic sites and single-strand breaks in cellular DNA. In the present study, an attempt has been made to investigate the involvement of BER genes and proteins in the radio-adaptive response in human resting peripheral blood mononuclear cells (PBMC). Venous blood samples were collected from 20 randomly selected healthy male individuals with written informed consent. PBMC were isolated and irradiated at a priming dose of 0.1 Gy followed 4h later with a challenging dose of 2.0 Gy (primed cells). Quantitation of DNA damage was done using the alkaline comet assay immediately and expression profile of BER genes and proteins were studied 30 min after the challenging dose using real-time quantitative polymerase chain reaction and western blot, respectively. The overall result showed significant (P ≤ 0.05) reduction of DNA damage in terms of percentage of DNA in tail (%T) with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4 h. Twelve individuals showed significant (P ≤ 0.05) reduction in %T whereas eight individuals showed marginal reduction in DNA damage that was not statistically significant. However, at the transcriptional level, BER genes such as APE1, FEN1 and LIGASE1 showed significant (P ≤ 0.05) up-regulation in both groups. Significant (P ≤ 0.05) up-regulation was also observed at the protein level for OGG1, APE1, MBD4, FEN1 and LIGASE1 in primed cells. Up-regulation of some BER genes and proteins such as APE1, FEN1 and LIGASE1 in primed cells of resting PBMC is suggestive of active involvement of the BER pathway in radio-adaptive response

  4. Quantum mechanical study of the β- and δ-lyase reactions during the base excision repair process: application to FPG.

    PubMed

    Sowlati-Hashjin, Shahin; Wetmore, Stacey D

    2015-10-14

    Bacterial FPG (or MutM) is a bifunctional DNA glycosylase that is primarily responsible for excising 8-oxoguanine (OG) from the genome by cleaving the glycosidic bond and the DNA backbone at the 3'- and 5'-phosphates of the damaged nucleoside. In the present work, quantum mechanical methods (SMD-M06-2X/6-311+G(2df,2p)//IEF-PCM-B3LYP/6-31G(d)) and a ring-opened Schiff base model that includes both the 3'- and 5'-phosphate groups are used to investigate the β- and δ-elimination reactions facilitated by FPG. Both the β- and δ-elimination reactions are shown to proceed through an E1cB mechanism that involves proton abstraction prior to the phosphate-ribose bond cleavage. Since transition states for the phosphate elimination reactions could not be characterized in the absence of leaving group protonation, our work confirms that the phosphate elimination reactions require protonation by a residue in the FPG active site, and can likely be further activated by additional active-site interactions. Furthermore, our model suggests that 5'-PO4 activation may proceed through a nearly isoenergetic direct (intramolecular) proton transfer involving the O4' proton of the deoxyribose of the damaged nucleoside. Regardless, our model predicts that both 3'- and 5'-phosphate protonation and elimination steps occur in a concerted reaction. Most importantly, our calculated barriers for the phosphate cleavage reactions reveal inherent differences between the β- and δ-elimination steps. Indeed, our calculations provide a plausible explanation for why the δ-elimination rather than the β-elimination is the rate-determining step in the BER facilitated by FPG, and why some bifunctional glycosylases (including the human counterpart, hOgg1) lack δ-lyase activity. Together, the new mechanistic features revealed by our work can be used in future large-scale modeling of the DNA-protein system to unveil the roles of key active sites residues in these relatively unexplored BER steps.

  5. The effects of 1-nitropyrene on oxidative DNA damage and expression of DNA repair enzymes.

    PubMed

    Kim, Yong-Dae; Ko, Young-Jun; Kawamoto, Toshihiro; Kim, Heon

    2005-05-01

    Nitropyrenes (NPs) present in diesel and gasoline emissions are mutagenic and carcinogenic in experimental animals. Nitro-reduction of 1-NP causes oxidative stress. It is unclear whether 8-hydroxydeoxyguanosine (8-OH-dG) is produced from 1-NP and whether it contributes to the carcinogenic activity of 1-NP. In this study, we measured the level of reactive oxygen species (ROS) in cultured human lung epithelial cells after exposure to 1-NP and the intracellular level of 8-OH-dG and expression level of the 8-OH-dG repair enzymes. As results, 1-NP induced the generation of 8-OH-dG via ROS, but 8-OH-dG repair enzymes prevented an increase of 8-OH-dG formation in cellular DNA of the A549 cell line below 250 microM of 1-NP. These data suggest that 1-NP can induce oxidative DNA damage by generation of ROS, which may play a role in the carcinogenesis induced by 1-NP. These data also suggest that individuals with impaired DNA repair enzymes might be more susceptible to lung cancer induced by 1-NP. PMID:15953848

  6. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  7. Influence of XPB helicase on recruitment and redistribution of nucleotide excision repair proteins at sites of UV-induced DNA damage.

    PubMed

    Oh, Kyu-Seon; Imoto, Kyoko; Boyle, Jennifer; Khan, Sikandar G; Kraemer, Kenneth H

    2007-09-01

    The XPB DNA helicase, a subunit of the basal transcription factor TFIIH, is also involved in nucleotide excision repair (NER). We examined recruitment of NER proteins in XP-B cells from patients with mild or severe xeroderma pigmentosum (XP) having different XPB mutations using local UV-irradiation through filters with 5 microm pores combined with fluorescent antibody labeling. XPC was rapidly recruited to UV damage sites containing DNA photoproducts (cyclobutane pyrimidine dimers, CPD) in all the XP-B and normal cells, thus reflecting its role in damage recognition prior to the function of XPB. Cells from the mild XP-B patients, with a missense mutation, showed delayed recruitment of all NER proteins except XPC to UV damage sites, demonstrating that this mutation impaired localization of these proteins. Surprisingly, in cells from severely affected patients, with a C-terminal XPB mutation, XPG and XPA proteins were normally recruited to UV damage sites demonstrating that this mutation permits recruitment of XPG and XPA. In marked contrast, in all the XP-B cells recruitment of XPF was absent immediately after UV and was delayed by 0.5 and 3 h in cells from the mild and severely affected XP patients, respectively. Redistribution of NER proteins was nearly complete in normal cells by 3 h but by 24 h redistribution was only partially present in cells from mild patients and virtually absent in cells from the severely affected patients. Ineffectual repair of UV-induced photoproducts resulting from delayed recruitment and impaired redistribution of NER proteins may contribute to the markedly increased frequency of skin cancer in XP patients. PMID:17509950

  8. Influence of XPB helicase on recruitment and redistribution of nucleotide excision repair proteins at sites of UV-induced DNA damage.

    PubMed

    Oh, Kyu-Seon; Imoto, Kyoko; Boyle, Jennifer; Khan, Sikandar G; Kraemer, Kenneth H

    2007-09-01

    The XPB DNA helicase, a subunit of the basal transcription factor TFIIH, is also involved in nucleotide excision repair (NER). We examined recruitment of NER proteins in XP-B cells from patients with mild or severe xeroderma pigmentosum (XP) having different XPB mutations using local UV-irradiation through filters with 5 microm pores combined with fluorescent antibody labeling. XPC was rapidly recruited to UV damage sites containing DNA photoproducts (cyclobutane pyrimidine dimers, CPD) in all the XP-B and normal cells, thus reflecting its role in damage recognition prior to the function of XPB. Cells from the mild XP-B patients, with a missense mutation, showed delayed recruitment of all NER proteins except XPC to UV damage sites, demonstrating that this mutation impaired localization of these proteins. Surprisingly, in cells from severely affected patients, with a C-terminal XPB mutation, XPG and XPA proteins were normally recruited to UV damage sites demonstrating that this mutation permits recruitment of XPG and XPA. In marked contrast, in all the XP-B cells recruitment of XPF was absent immediately after UV and was delayed by 0.5 and 3 h in cells from the mild and severely affected XP patients, respectively. Redistribution of NER proteins was nearly complete in normal cells by 3 h but by 24 h redistribution was only partially present in cells from mild patients and virtually absent in cells from the severely affected patients. Ineffectual repair of UV-induced photoproducts resulting from delayed recruitment and impaired redistribution of NER proteins may contribute to the markedly increased frequency of skin cancer in XP patients.

  9. Lack of association between polymorphisms of the DNA base excision repair genes MUTYH and hOGG1 and keratoconus in a Polish subpopulation

    PubMed Central

    Synowiec, Ewelina; Wójcik, Katarzyna A.; Czubatka, Anna; Polakowski, Piotr; Izdebska, Justyna; Szaflik, Jerzy; Błasiak, Janusz

    2015-01-01

    Introduction Keratoconus (KC) is a non-inflammatory thinning of the cornea and a leading indication for corneal transplantation. Oxidative stress plays a role in the pathogenesis of this disease. The products of the hOGG1 and MUTYH genes play an important role in the repair of oxidatively modified DNA in the base excision repair pathway. We hypothesized that variability in these genes may change susceptibility to oxidative stress and predispose individuals to the development of KC. We investigated the possible association between the c.977C>G polymorphism of the hOGG1 gene (rs1052133) and the c.972G>C polymorphism of the MUTYH gene (rs3219489) and KC occurrence as well as the modulation of this association by some KC risk factors. Material and methods A total of 205 patients with KC and 220 controls were included in this study. The polymorphisms were genotyped with polymerase chain reaction (PCR) restriction fragment length polymorphism and PCR-confronting two-pair primer techniques. Differences in genotype and allele frequency distributions were evaluated using the χ2 test, and KC risk was estimated with an unconditional multiple logistic regression with and without adjustment for co-occurrence of visual impairment, allergies, sex and family history for KC. Results We did not find any association between the genotypes and combined genotypes of the c.977C>G polymorphism of the hOGG1 gene and the c.972G>C polymorphism of the MUTYH gene and the occurrence of KC. Conclusions Our findings suggest that the c.977C>G-hOGG1 polymorphism and the c.972G>C-MUTYH polymorphism may not be linked with KC occurrence in this Polish subpopulation. PMID:26528356

  10. Repair of oxidative DNA damage is delayed in the Ser326Cys polymorphic variant of the base excision repair protein OGG1.

    PubMed

    Kershaw, Rachael M; Hodges, Nikolas J

    2012-07-01

    Gene-environment interactions influence an individual's risk of disease development. A common human 8-oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326-hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated mOgg1 (-/-) null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 hOGG1 allele.

  11. Crosstalk between MSH2–MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair

    PubMed Central

    Lai, Yanhao; Budworth, Helen; Beaver, Jill M.; Chan, Nelson L. S.; Zhang, Zunzhen; McMurray, Cynthia T.; Liu, Yuan

    2016-01-01

    Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion. PMID:27546332

  12. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  13. Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels.

    PubMed

    Mukherjee, Bidisha; Bindhani, Banani; Saha, Hirak; Ray, Manas Ranjan

    2014-09-01

    To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age.

  14. Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels.

    PubMed

    Mukherjee, Bidisha; Bindhani, Banani; Saha, Hirak; Ray, Manas Ranjan

    2014-09-01

    To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age. PMID:25128766

  15. Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

    PubMed

    Nakazawa, Yuka; Sasaki, Kensaku; Mitsutake, Norisato; Matsuse, Michiko; Shimada, Mayuko; Nardo, Tiziana; Takahashi, Yoshito; Ohyama, Kaname; Ito, Kosei; Mishima, Hiroyuki; Nomura, Masayo; Kinoshita, Akira; Ono, Shinji; Takenaka, Katsuya; Masuyama, Ritsuko; Kudo, Takashi; Slor, Hanoch; Utani, Atsushi; Tateishi, Satoshi; Yamashita, Shunichi; Stefanini, Miria; Lehmann, Alan R; Yoshiura, Koh-ichiro; Ogi, Tomoo

    2012-05-01

    UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders. PMID:22466610

  16. Frequent Imprecise Excision among Reversions of a P Element-Caused Lethal Mutation in Drosophila

    PubMed Central

    Voelker, Robert A.; Greenleaf, Arno L.; Gyurkovics, Henrik; Wisely, G. Bruce; Huang, Shu-Mei; Searles, Lillie L.

    1984-01-01

    RpII215D50 (= D50) is a lethal mutation caused by the insertion of a 1.3-kb P element 5' to sequences encoding the largest (215 kilodaltons) subunit of Drosophila RNA polymerase II. In dysgenic males D50 reverted to nonlethality at frequencies ranging from 2.6 to 6.5%. These reversions resulted from loss of P element sequences. Genetic tests of function and restriction enzyme analysis of revertant DNAs revealed that 35% or more of the reversion events were imprecise excisions. Two meiotic mutations that perturb excision repair and postreplication repair (mei-9a and mei-41D5, respectively) had no influence on reversion frequency but may have increased the proportion of imprecise excisions. We suggest that these excisions are by-products of, rather than intermediates in, the transposition process. PMID:17246216

  17. An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts

    PubMed Central

    Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam

    2013-01-01

    Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095

  18. Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC

    PubMed Central

    Forestier, Anne; Douki, Thierry; De Rosa, Viviana; Béal, David; Rachidi, Walid

    2015-01-01

    Repair of oxidative DNA damage, particularly Base Excision Repair (BER), impairment is often associated with Alzheimer’s disease pathology. Here, we aimed at investigating the complete Nucleotide Excision Repair (NER), a DNA repair pathway involved in the removal of bulky DNA adducts, status in an Alzheimer-like cell line. The level of DNA damage was quantified using mass spectrometry, NER gene expression was assessed by qPCR, and the NER protein activity was analysed through a modified version of the COMET assay. Interestingly, we found that in the presence of the Amyloid β peptide (Aβ), NER factors were upregulated at the mRNA level and that NER capacities were also specifically increased following oxidative stress. Surprisingly, NER capacities were not differentially improved following a typical NER-triggering of ultraviolet C (UVC) stress. Oxidative stress generates a differential and specific DNA damage response in the presence of Aβ. We hypothesized that the release of NER components such as DNA damage binding protein 2 (DDB2) and Xeroderma Pigmentosum complementation group C protein (XPC) following oxidative stress might putatively involve their apoptotic role rather than DNA repair function. PMID:26263968

  19. Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

    PubMed

    Stohl, Elizabeth A; Seifert, H Steven

    2006-11-01

    The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2). PMID:16936020

  20. Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase

    SciTech Connect

    Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M.

    2010-08-12

    Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

  1. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    PubMed

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón Y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; Swe-Brca; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I; Beattie, Mary S; Domchek, Susan M; Nathanson, Katherine; Rebbeck, Timothy R; Arun, Banu K; Karlan, Beth Y; Walsh, Christine; Lester, Jenny; John, Esther M; Whittemore, Alice S; Daly, Mary B; Southey, Melissa; Hopper, John; Terry, Mary B; Buys, Saundra S; Janavicius, Ramunas; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Steele, Linda; Neuhausen, Susan L; Ding, Yuan Chun; Hansen, Thomas V O; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A; van Os, Theo A M; van der Kolk, Lizet; de Lange, J L; Meijers-Heijboer, Hanne E J; van der Hout, A H; van Asperen, Christi J; Gómez Garcia, Encarna B; Hoogerbrugge, Nicoline; Collée, J Margriet; van Deurzen, Carolien H M; van der Luijt, Rob B; Devilee, Peter; Hebon; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R; Healey, Sue; Investigators, Kconfab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F; Offit, Kenneth; Couch, Fergus J; Chenevix-Trench, Georgia; Antoniou, Antonis C; Benitez, Javier

    2014-04-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  2. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    PubMed Central

    Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; SWE-BRCA; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gómez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collée, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; HEBON; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th.; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Investigators, kConFab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10−3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10−3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. PMID:24698998

  3. The association of six polymorphisms of five genes involved in three steps of nucleotide excision repair pathways with hepatocellular cancer risk

    PubMed Central

    Yang, Huai-wei; Sun, Li-ping; Yuan, Yuan

    2016-01-01

    Background Hundreds of single nucleotide polymorphisms (SNPs) of the genes encoding nucleotide excision repair (NER) proteins are involved in every step of the DNA recognition–unwinding–incision process, which may affect cancer risk. However, only a limited number of studies have examined the association of NER SNPs with hepatocellular cancer (HCC) risk. Results In screening stage, single-locus analysis showed that six SNPs in five genes were associated with HCC risk, including three risk SNPs (XPA rs10817938, XPC rs1870134 and ERCC2 rs238417) and three protective SNPs (ERCC1 rs2298881 and rs3212961, and ERCC5 rs873601). In verification stage, only XPC rs1870134 was verified to be associated with HCC risk (P = 4.7 × 10−4). Furthermore, multivariate logistic regression and MDR analysis consistently revealed a gene–gene interaction among ERCC1 rs2298881 and XPC rs1870134 SNPs associated with HCC risk (Pinteraction = 0.023). When analyzing the effect of the positive SNP on the mRNA expression, we found XPC rs1870134 GG genotype which was associated with an increased HCC risk showed lower XPC mRNA expression. Methods This study designed as “screening-verification” experiments and included a total of 1472 participants (570 HCC patients vs. 902 controls). We explored 39 SNPs in eight genes involved in NER Pathways, including XPA, XPC, DDB2, ERCC3, ERCC2, ERCC1, ERCC4 and ERCC5, using Sequenom MassARRAY and KASPar platform. Eighty-six cases of HCC and the neighboring noncancerous tissues were subjected to the measurement of mRNA expression level of the promising gene. Conclusions XPC promoter rs1870134 SNP and SNP-SNP interaction were associated with HCC risk. PMID:26967386

  4. Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

    PubMed

    Guilliam, Thomas A; Keen, Benjamin A; Brissett, Nigel C; Doherty, Aidan J

    2015-08-18

    Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here.

  5. Breast cancer resistance protein (BCRP) and excision repair cross complement-1 (ERCC1) expression in esophageal cancers and response to cisplatin and irinotecan based chemotherapy

    PubMed Central

    Bharthuar, Anubha; Black, Jennifer D.; Levea, Charles; Malhotra, Usha; Mashtare, Terry L.; Iyer, Renuka

    2014-01-01

    Background Esophageal cancer patients face a dismal outcome despite tri-modality management and median survival remains 15-18 months. Breast cancer resistance protein (BCRP) is an ATP-dependent efflux protein associated with chemotherapy resistance. The role of BCRP expression in esophageal cancer and normal esophageal cells is not known. Excision repair cross complement-1 (ERCC1) overexpression has been correlated with poorer response to cisplatin based chemotherapy. We examined the expression of BCRP and ERCC1 in patients with esophageal cancer and correlated it with survival in patients receiving irinotecan and cisplatin based chemotherapy. Methods With IRB approval, 40 cases of esophageal cancer diagnosed from 2004-2008, were stained for BCRP and ERCC1 expression by immunohistochemistry and scored by a pathologist blinded to clinical data. Baseline demographics, therapy given and survival data were collected and correlated with BCRP and ERCC1 expression. Fisher’s exact test was used to determine association between BCRP and ERCC1 expression and demographics. Cox proportional hazards model was used for association of BCRP and ERCC1 with survival. Results On immunohistochemistry, 30/40 cancers (75%) expressed BCRP. Interestingly, down-regulation of BCRP expression in tumor compared with normal cells was seen in 40% of patients. ERCC1 positivity was seen in 15/30 cases (50%). Median overall survival (OS) was 19 months with no difference in survival between BCRP positive and negative patients (P=0.13) or ERCC1 positive and negative patients (P=0.85). Estimated hazard ratio (HR) of death for BRCP positive patients was 2.29 (95% CI: 0.79-6.64) and for ERCC1 positive patients was 1.09 (95% CI: 0.46-2.56). There was no association of BCRP and ERCC1 expression with disease stage, age, gender or histology. For patients who received cisplatin and irinotecan as first line chemotherapy, there was no difference in survival based on BCRP or ERCC1 status. Conclusions BCRP

  6. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

    SciTech Connect

    Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

    2013-03-22

    Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

  7. Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme.

    PubMed

    Yarosh, D; Klein, J; Kibitel, J; Alas, L; O'Connor, A; Cummings, B; Grob, D; Gerstein, D; Gilchrest, B A; Ichihashi, M; Ogoshi, M; Ueda, M; Fernandez, V; Chadwick, C; Potten, C S; Proby, C M; Young, A R; Hawk, J L

    1996-06-01

    Xeroderma pigmentosum (XP) is a rare genetic disease in which patients are defective in DNA repair and are extremely sensitive to solar UV radiation exposure. A new treatment approach was tested in these patients, in which a prokaryotic DNA repair enzyme specific for UV-induced DNA damage was delivered into the skin by means of topically applied liposomes to supplement the deficient activity. Acute and chronic safety testing in both mice and humans showed neither adverse reactions nor significant changes in serum chemistry or in skin histology. The skin of XP patients treated with the DNA repair liposomes had fewer cyclobutylpyrimidine dimers in DNA and showed less erythema than did control sites. The results encourage further clinical testing of this new enzyme therapy approach.

  8. Downregulation of the DNA repair enzyme apurinic/apyrimidinic endonuclease 1 stimulates transforming growth factor-β1 production and promotes actin rearrangement.

    PubMed

    Sakai, Yuri; Yamamori, Tohru; Yasui, Hironobu; Inanami, Osamu

    2015-05-22

    The DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in base excision repair and functions as a reductive activator of various transcription factors. Multiple other functionalities have been ascribed to APE1 in addition to these major functions. A recent study showed that APE1 knockdown upregulated the expression of a set of genes related to extracellular matrix (ECM) production, indicating an additional novel biological role for this enzyme. Based on this finding, we have investigated the effect of APE1 downregulation on ECM-related gene expression and its biological consequences. Endogenous APE1 expression was downregulated in human cervical carcinoma HeLa cells and human lung carcinoma A549 cells using siRNA. When the expression of six ECM-related genes (TGFB1, LAMC1, FN1, COL1A1, COL3A1, and COL4A1) was evaluated, we found that APE1 knockdown upregulated the expression of TGFB1 in both cell lines. APE1 downregulation promoted actin rearrangement, inducing F-actin accumulation in HeLa cells and the dissipation of stress fibers in A549 cells. We also discovered that APE1 knockdown enhanced cellular motility in A549 cells, which was suppressed by the inhibition of transforming growth factor (TGF)-β1 signaling. These results suggested that APE1 controls the organization of actin cytoskeleton through the regulation of TGF-β1 expression, providing novel insights into the biological significance of APE1. PMID:25858321

  9. Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

    PubMed Central

    Stege, Helger; Roza, Len; Vink, Arie A.; Grewe, Markus; Ruzicka, Thomas; Grether-Beck, Susanne; Krutmann, Jean

    2000-01-01

    Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection. PMID:10660687

  10. UV-induced DNA damage and repair: a review.

    PubMed

    Sinha, Rajeshwar P; Häder, Donat P

    2002-04-01

    Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms. DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence Isomers. However, cells have developed a number of repair or tolerance mechanism to counteract the DNA damage caused by UV or any other stressors. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.

  11. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  12. Quantum mechanics/molecular mechanics study on the oxygen binding and substrate hydroxylation step in AlkB repair enzymes.

    PubMed

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N(1) -methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)-oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ- and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.

  13. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  14. Inducible nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers in the cell cycle of the budding yeast Saccharomyces cerevisiae: evidence that inducible NER is confined to the G1 phase of the mitotic cell cycle.

    PubMed

    Scott, A D; Waters, R

    1997-03-18

    We previously reported on an inducible component of nucleotide excision repair in Saccharomyces cerevisiae that is controlled by the RAD16 gene. Here we describe a study of this event at the MAT alpha and HML alpha mating-type loci and on the transcribed (TS) and nontranscribed (NTS) strands of the RAD16 gene. Events were examined at various stages of the mitotic cycle in cells synchronised by centrifugal elutriation. Repair of cyclobutane pyrimidine dimers (CPDs) following a single UV dose does not vary significantly in different stages of the mitotic cell cycle. CPDs are removed more rapidly from the transcriptionally active MAT alpha locus than from the silent HML alpha locus, and the TS of RAD16 is repaired faster than the NTS in all stages of the cycle following a single UV irradiation. Enhanced excision of CPDs at MAT alpha and HML alpha can be induced only in the G1 and early S stages of the cell cycle. Here prior irradiation of cells with 25 J/m2 enhances the removal of CPDs following a second UV dose of 70 J/m2. The level of enhancement of repair does not differ significantly between MAT alpha and HML alpha in G1. Enhanced removal of CPDs is absent when cells receive the inducing dose in late S or G2/M. Repair of CPDs in both strands of RAD16 is similarly enhanced only if cells receive the initial irradiation in G1 and early S. The level of enhanced removal of CPDs is not significantly different in the TS and NTS of RAD16 either in asynchronous cells or in cells preirradiated in G1 and early S. It has been shown by others that UV-induced expression of RAD16 remains at high levels if cells are held in G1 by treatment with alpha factor. Therefore the increase in RAD16 transcript levels in G1 may be responsible for the ability to enhance NER solely in this stage of the cell cycle.

  15. [Retarded excision of pyrimidine dimers in human unstimulated lymphocytes].

    PubMed

    Snopov, S A; Roza, L; de Gruijl, F R

    2006-01-01

    Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.

  16. Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

    PubMed Central

    Fu, Qiong; Chow, Julia; Bernstein, Kara A.; Makharashvili, Nodar; Arora, Sucheta; Lee, Chia-Fang; Person, Maria D.; Rothstein, Rodney

    2014-01-01

    In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state. PMID:24344201

  17. Functional Polymorphisms of Base Excision Repair Genes XRCC1 and APEX1 Predict Risk of Radiation Pneumonitis in Patients With Non-Small Cell Lung Cancer Treated With Definitive Radiation Therapy

    SciTech Connect

    Yin Ming; Liao Zhongxing; Liu Zhensheng; Wang, Li-E; Gomez, Daniel; Komaki, Ritsuko; Wei Qingyi

    2011-11-01

    Purpose: To explore whether functional single nucleotide polymorphisms (SNPs) of base-excision repair genes are predictors of radiation treatment-related pneumonitis (RP), we investigated associations between functional SNPs of ADPRT, APEX1, and XRCC1 and RP development. Methods and Materials: We genotyped SNPs of ADPRT (rs1136410 [V762A]), XRCC1 (rs1799782 [R194W], rs25489 [R280H], and rs25487 [Q399R]), and APEX1 (rs1130409 [D148E]) in 165 patients with non-small cell lung cancer (NSCLC) who received definitive chemoradiation therapy. Results were assessed by both Logistic and Cox regression models for RP risk. Kaplan-Meier curves were generated for the cumulative RP probability by the genotypes. Results: We found that SNPs of XRCC1 Q399R and APEX1 D148E each had a significant effect on the development of Grade {>=}2 RP (XRCC1: AA vs. GG, adjusted hazard ratio [HR] = 0.48, 95% confidence interval [CI], 0.24-0.97; APEX1: GG vs. TT, adjusted HR = 3.61, 95% CI, 1.64-7.93) in an allele-dose response manner (Trend tests: p = 0.040 and 0.001, respectively). The number of the combined protective XRCC1 A and APEX1 T alleles (from 0 to 4) also showed a significant trend of predicting RP risk (p = 0.001). Conclusions: SNPs of the base-excision repair genes may be biomarkers for susceptibility to RP. Larger prospective studies are needed to validate our findings.

  18. Novel mechanism of regulation of the DNA repair enzyme OGG1 in tuberin-deficient cells

    PubMed Central

    Habib, Samy L.; Bhandari, Besant K.; Sadek, Nahed; Abboud-Werner, Sherry L.; Abboud, Hanna E.

    2010-01-01

    Tuberin (protein encodes by tuberous sclerosis complex 2, Tsc2) deficiency is associated with the decrease in the DNA repair enzyme 8-oxoG-DNA glycosylase (OGG1) in tumour kidney of tuberous sclerosis complex (TSC) patients. The purpose of this study was to elucidate the mechanisms by which tuberin regulates OGG1. The partial deficiency in tuberin expression that occurs in the renal proximal tubular cells and kidney cortex of the Eker rat is associated with decreased activator protein 4 (AP4) and OGG1 expression. A complete deficiency in tuberin is associated with loss of AP4 and OGG1 expression in kidney tumour from Eker rats and the accumulation of significant levels of 8-oxo-deoxyguanosine. Knockdown of tuberin expression in human renal epithelial cells (HEK293) with small interfering RNA (siRNA) also resulted in a marked decrease in the expression of AP4 and OGG1. In contrast, overexpression of tuberin in HEK293 cells increased the expression of AP4 and OGG1 proteins. Downregulation of AP4 expression using siRNA resulted in a significant decrease in the protein expression of OGG1. Immunoprecipitation studies show that AP4 is associated with tuberin in cells. Gel shift analysis and chromatin immunoprecipitation identified the transcription factor AP4 as a positive regulator of the OGG1 promoter. AP4 DNA-binding activity is significantly reduced in Tsc2−/− as compared with Tsc2+/+ cells. Transcriptional activity of the OGG1 promoter is also decreased in tuberin-null cells compared with wild-type cells. These data indicate a novel role for tuberin in the regulation of OGG1 through the transcription factor AP4. This regulation may be important in the pathogenesis of kidney tumours in patients with TSC disease. PMID:20837600

  19. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    NASA Astrophysics Data System (ADS)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  20. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Parsons, Zachary D; Yuen, Philip K; David, Sheila S; Igarashi, Yasuhiro; Eichman, Brandt F

    2015-11-12

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision. PMID:26524531

  1. An electrochemiluminescence biosensor for 8-oxo-7,8-dihydro-2'-deoxyguanosine quantification and DNA repair enzyme activity analysis using a novel bifunctional probe.

    PubMed

    Wu, Yiping; Yang, Xiqiang; Zhang, Bintian; Guo, Liang-Hong

    2015-07-15

    A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0U/µL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg(2+) and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04µM and 4.34nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg.

  2. Molecular mechanisms of DNA repair inhibition by caffeine

    SciTech Connect

    Selby, C.P.; Sancar, A. )

    1990-05-01

    Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

  3. Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes.

    PubMed

    Amundsen, Susan K; Spicer, Timothy; Karabulut, Ahmet C; Londoño, Luz Marina; Eberhart, Christina; Fernandez Vega, Virneliz; Bannister, Thomas D; Hodder, Peter; Smith, Gerald R

    2012-05-18

    The AddAB and RecBCD helicase-nucleases are related enzymes prevalent among bacteria but not eukaryotes and are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although these enzymes have been extensively studied both genetically and biochemically, inhibitors specific for this class of enzymes have not been reported. We developed a high-throughput screen based on the ability of phage T4 gene 2 mutants to grow in Escherichia coli only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized this screen for use in 1536-well plates and screened 326,100 small molecules in the NIH molecular libraries sample collection for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli recBCD deletion strain. Secondary screening used assays with cells expressing AddAB or RecBCD and a viability assay that measured the effect of compounds on cell growth without phage infection. From this screening campaign, 12 compounds exhibiting efficacy and selectivity were tested for inhibition of purified AddAB and RecBCD helicase and nuclease activities and in cell-based assays for recombination; seven were active in the 0.1-50 μM range in one or another assay. Compounds structurally related to two of these were similarly tested, and three were active in the 0.1-50 μM range. These compounds should be useful in further enzymatic, genetic, and physiological studies of these enzymes, both purified and in cells. They may also lead to useful antibacterial agents, since this class of enzymes is needed for successful bacterial infection of mammals.

  4. Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker.

    PubMed

    Havran, Ludek; Vacek, Jan; Cahová, Katerina; Fojta, Miroslav

    2008-07-01

    This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.

  5. Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain.

    PubMed

    Schaaper, R M; Dunn, R L; Glickman, B W

    1987-11-20

    We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base

  6. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair.

    PubMed

    Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Wani, Altaf

    2012-12-01

    Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER. PMID:23083890

  7. Staphylococcus aureus Sepsis and Mitochondrial Accrual of the 8-Oxoguanine DNA Glycosylase DNA Repair Enzyme in Mice

    PubMed Central

    Bartz, Raquel R.; Suliman, Hagir B.; Fu, Ping; Welty-Wolf, Karen; Carraway, Martha Sue; MacGarvey, Nancy Chou; Withers, Crystal M.; Sweeney, Timothy E.; Piantadosi, Claude A.

    2011-01-01

    Rationale: Damage to mitochondrial DNA (mtDNA) by the production of reactive oxygen species during inflammatory states, such as sepsis, is repaired by poorly understood mechanisms. Objectives: To test the hypothesis that the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), contributes to mtDNA repair in sepsis. Methods: Using a well-characterized mouse model of Staphylococcus aureus sepsis, we analyzed molecular markers for mitochondrial biogenesis and OGG1 translocation into liver mitochondria as well as OGG1 mRNA expression at 0, 24, 48, and 72 hours after infection. The effects of OGG1 RNA silencing on mtDNA content were determined in control, tumor necrosis factor-α, and peptidoglycan-exposed rat hepatoma cells. Based on in situ analysis of the OGG1 promoter region, chromatin immunoprecipitation assays were performed for nuclear respiratory factor (NRF)-1 and NRF-2α GA-binding protein (GABP) binding to the promoter of OGG1. Measurements and Main Results: Mice infected with 107 cfu S. aureus intraperitoneally demonstrated hepatic oxidative mtDNA damage and significantly lower hepatic mtDNA content as well as increased mitochondrial OGG1 protein and enzyme activity compared with control mice. The infection also caused increases in hepatic OGG1 transcript levels and NRF-1 and NRF-2α transcript and protein levels. A bioinformatics analysis of the Ogg1 gene locus identified several promoter sites containing NRF-1 and NRF-2α DNA binding motifs, and chromatin immunoprecipitation assays confirmed in situ binding of both transcription factors to the Ogg1 promoter within 24 hours of infection. Conclusions: These studies identify OGG1 as an early mitochondrial response protein during sepsis under regulation by the NRF-1 and NRF-2α transcription factors that regulate mitochondrial biogenesis. PMID:20732986

  8. Arsenic is cytotoxic at micromolar concentration, but does not inhibit purified human DNA repair enzymes at less than millimolar concentrations

    SciTech Connect

    Su, Lin; Hu, Yu; Dunlop, B.

    1997-10-01

    Arsenic is a well-known human carcinogen, but not a mutagen. However it can act as a co-mutagen with UV and alkylating agents, and has been shown to inhibit DNA repair. The activities of several purified human enzymes involved in DNA repair have been tested in the presence of inorganic arsenite [As(III)] and arsenate [As(V)]. We have not found that both As(III) and As(V) stimulated the activity of DNA polymerase {beta} (pol {beta}), O{sup 6}methylguanine DNA methyltransferase (MGMT), and DNA ligase III. The activity of pol {beta} was increased up to 3.5-fold in the presence of 50 mM As (III), and 2-fold in the presence of 20 mM As(V). Inhibition of enzyme activity was only observed with concentrations of As(III) and As(V) higher than 100 mM. Terminal deoxynucleotidal transferase (TdT), an enzyme with homology to pol {beta}, is also stimulated 3-fold by 50 mM As(III). Unlike pol {beta} and TdT, MGMT was preferentially activated by millimolar As(V), rather than As(III). Similar concentrations of inorganic phosphate also increased the activity of MGMT. The activity of DNA ligase I was inhibited by 1 to 5 mM As(III). However, both DNA ligase I and DNA ligase III were significantly activated by As(V). In contrast to these results, human keratinocyte cells exhibit significant cytotoxicity when exposed to 10 {mu}M As(III) and 200 {mu}M AS(V). Cell survival was decreased by over 50% at these concentrations, as measured by neutral red uptake, LDH release, and MTT uptake. Interestingly, both As(III) and As(V) produced increased cell proliferation at submicromolar concentrations. These results suggest that arsenic compounds do not exert their toxic effects by direct inhibition of DNA repair enzymes, but by other mechanisms.

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

    PubMed Central

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

    1992-01-01

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

  10. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    PubMed Central

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  11. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

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

    PubMed Central

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

    2015-01-01

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

  13. hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway

    PubMed Central

    Paquet, Nicolas; Adams, Mark N.; Leong, Vincent; Ashton, Nicholas W.; Touma, Christine; Gamsjaeger, Roland; Cubeddu, Liza; Beard, Sam; Burgess, Joshua T.; Bolderson, Emma; O'Byrne, Ken J.; Richard, Derek J.

    2015-01-01

    The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome. PMID:26261212

  14. Loss of expression of DNA repair enzyme MGMT in oral leukoplakia and early oral squamous cell carcinoma. A prognostic tool?

    PubMed

    Rodríguez, María J; Acha, Amelia; Ruesga, María T; Rodríguez, Carlos; Rivera, José M; Aguirre, José M

    2007-01-01

    MGMT is a specific DNA repair enzyme that removes alkylating lesions and therefore plays an important role in maintaining normal cell physiology and genomic stability. Loss of expression of MGMT is associated with increased carcinogenic risk and sensitivity to methylating agents in different types of tumours. The expression of MGMT was immunohistochemically assessed in 12 normal oral mucosa, 38 oral leukoplakias and 33 early oral squamous cell carcinomas. The results were correlated with clinicopathological data. We found a significant loss of MGMT protein expression from leukoplakia when compared with early squamous cell carcinoma. We also observed a statistically significant relationship between smoking and the loss of MGMT protein expression. Loss of MGMT expression could be considered an early event in oral carcinogenesis with possible prognostic implications.

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

    PubMed Central

    Fleck, O; Michael, H; Heim, L

    1992-01-01

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

  16. Predictive and Prognostic Value of Ribonucleotide Reductase Regulatory Subunit M1 and Excision Repair Cross-Complementation Group 1 in Advanced Urothelial Carcinoma (UC) Treated with First-Line Gemcitabine Plus Platinum Combination Chemotherapy.

    PubMed

    Kim, Miso; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe; Lee, Eunsik; Keam, Bhumsuk; Kim, Tae Min; Heo, Dae Seog; Lee, Se-Hoon; Moon, Kyung Chul

    2015-01-01

    Preclinical and clinical studies have suggested that expression of ribonucleotide reductase regulatory subunit M1 (RRM1) and excision repair cross-complementation group 1 (ERCC1) is associated with resistance to gemcitabine and cisplatin, respectively. We evaluated the significance of RRM1 and ERCC1 expression to predict tumor response to gemcitabine plus platinum chemotherapy (GP) and survival in advanced UC. We retrospectively collected tumor samples and reviewed clinical data of 53 patients with unresectable or metastatic UC, who were treated with first-line GP. RRM1 and ERCC1 expression were measured by immunohistochemistry. Among 53 patients, 12 (22.6%) and 26 (49.1%) patients had tumors that demonstrated a high expression for RRM1 and ERCC1, respectively. Twenty-nine (70.7%) of 41 patients with low RRM1 expression achieved a clinical response (complete + partial responses), but only 3 (25.0%) of 12 patients with high RRM1 expression achieved a clinical response after GP (P=0.007). Nineteen (70.4%) of 27 patients with low ERCC1 expression achieved a clinical response, while 13 (50.0%) of 26 patients with high ERCC1 expression achieved a clinical response (P=0.130). High RRM1 expression was associated with shorter progression free survival and overall survival (PFS P=0.006, OS P=0.006). Multivariate analysis confirmed that patients with high RRM1 expression had a significantly greater risk of progression and death than those with low RRM1 expression. ERCC1 status was not a significant predictor for PFS and OS. RRM1 expression was predictive and prognostic of clinical outcome in advanced UC treated with gemcitabine plus platinum combination chemotherapy.

  17. Construction and Analysis of Photolyase Mutants of Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of Photoreactivation, Nucleotide Excision Repair, and Mutagenic DNA Repair to Cell Survival and Mutability following Exposure to UV-B Radiation

    PubMed Central

    Kim, Jae J.; Sundin, George W.

    2001-01-01

    Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2. Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m−2 followed by a recovery period under photoreactivating conditions. A phr uvrA mutant of P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m−2. Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 using the cloned phr gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant rulAB. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing rulAB indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which rulAB only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 × 10−5 and 2.1 × 10−3, respectively, following a UV-B dose of 1,550 J m−2. The construction and characterization of phr mutants in the present study will facilitate the

  18. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus.

    PubMed

    Han, Jeonghoon; Won, Eun-Ji; Kim, Il-Chan; Yim, Joung Han; Lee, Su-Jae; Lee, Jae-Seong

    2014-10-01

    To examine the effects of gamma radiation on marine organisms, we irradiated several doses of gamma ray to the microzooplankton Brachionus koreanus, and measured in vivo and in vitro endpoints including the survival rate, lifespan, fecundity, population growth, gamma ray-induced oxidative stress, and modulated patterns of enzyme activities and gene expressions after DNA damage. After gamma radiation, no individuals showed any mortality within 96 h even at a high intensity (1200 Gy). However, a reduced fecundity (e.g. cumulated number of offspring) of B. koreanus at over 150 Gy was observed along with a slight decrease in lifespan. At 150 Gy and 200 Gy, the reduced fecundity of the rotifers led to a significant decrease in population growth, although in the second generation the population growth pattern was not affected even at 200 Gy when compared to the control group. At sub-lethal doses, reactive oxygen species (ROS) levels dose-dependently increased with GST enzyme activity. In addition, up-regulations of the antioxidant and chaperoning genes in response to gamma radiation were able to recover cellular damages, and life table parameters were significantly influenced, particularly with regard to fecundity. DNA repair-associated genes showed significantly up-regulated expression patterns in response to sublethal doses (150 and 200 Gy), as shown in the expression of the gamma-irradiated B. koreanus p53 gene, suggesting that these sublethal doses were not significantly fatal to B. koreanus but induced DNA damages leading to a decrease of the population size.

  19. Delineation of structural domains and identification of functionally important residues in DNA repair enzyme exonuclease VII

    PubMed Central

    Poleszak, Katarzyna; Kaminska, Katarzyna H.; Dunin-Horkawicz, Stanislaw; Lupas, Andrei; Skowronek, Krzysztof J.; Bujnicki, Janusz M.

    2012-01-01

    Exonuclease VII (ExoVII) is a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. We used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli, we determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit and residues D155, R205, H238 and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, we propose a model of sequence–structure–function relationships in ExoVII. PMID:22718974

  20. Crystal structure of E. coli endonuclease V, an essential enzyme for deamination repair

    PubMed Central

    Zhang, Zhemin; Jia, Qian; Zhou, Chun; Xie, Wei

    2015-01-01

    Endonuclease V (EndoV) is a ubiquitous protein present in all three kingdoms of life, responsible for the specific cleavages at the second phosphodiester bond 3’ to inosine. E. coli EndoV (EcEndoV) is the first member discovered in the EndoV family. It is a small protein with a compact gene organization, yet with a wide spectrum of substrate specificities. However, the structural basis of its substrate recognition is not well understood. In this study, we determined the 2.4 Å crystal structure of EcEndoV. The enzyme preserves the general ‘RNase H-like motif’ structure. Two subunits are almost fully resolved in the asymmetric unit, but they are not related by any 2-fold axes. Rather, they establish “head-to-shoulder” contacts with loose interactions between each other. Mutational studies show that mutations that disrupt the association mode of the two subunits also decrease the cleavage efficiencies of the enzyme. Further biochemical studies suggest that EcEndoV is able to bind to single-stranded, undamaged DNA substrates without sequence specificity, and forms two types of complexes in a metal-independent manner, which may explain the wide spectrum of substrate specificities of EcEndoV. PMID:26244280

  1. Dimer excision in Escherichia coli in the presence of caffeine

    SciTech Connect

    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 made and prolonged the time required to rejoin the discontinuities. Patch size, however, was unaffected by caffeine treatment.

  2. International congress on DNA damage and repair: Book of abstracts

    SciTech Connect

    Not Available

    1987-01-01

    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)

  3. Structures and activities of archaeal members of the LigD 3′-phosphoesterase DNA repair enzyme superfamily

    PubMed Central

    Smith, Paul; Nair, Pravin A.; Das, Ushati; Zhu, Hui; Shuman, Stewart

    2011-01-01

    LigD 3′-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3′-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis–à-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs—Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids)—and we report their atomic structures at 1.1 and 2.1 Å, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded β barrel and a 310 helix. Their active sites are located in a crescent-shaped groove on the barrel’s outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributed widely among taxa. PMID:21208981

  4. Structures and Activities of Archaeal Members of the LigD 3-Phosphoesterase DNA Repair Enzyme Superfamily

    SciTech Connect

    P Smith; P Nair; U Das; H Zhu; S Shuman

    2011-12-31

    LigD 3'-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3'-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis-a-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs - Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids) - and we report their atomic structures at 1.1 and 2.1 {angstrom}, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded {beta} barrel and a 3{sub 10} helix. Their active sites are located in a crescent-shaped groove on the barrel's outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributed widely among taxa.

  5. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

    PubMed Central

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  6. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

    PubMed

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  7. [A Nobel Prize for DNA repair].

    PubMed

    Jordan, Bertrand

    2016-01-01

    This year's Nobel Prize for chemistry recognizes the seminal contributions of three researchers who discovered the existence and the basic mechanisms of DNA repair: base excision repair, mismatch repair, and nucleotide excision repair. They have since been joined by many scientists elucidating diverse aspects of these complex mechanisms that now constitute a thriving research field with many applications, notably for understanding oncogenesis and devising more effective therapies. PMID:26850617

  8. [A Nobel Prize for DNA repair].

    PubMed

    Jordan, Bertrand

    2016-01-01

    This year's Nobel Prize for chemistry recognizes the seminal contributions of three researchers who discovered the existence and the basic mechanisms of DNA repair: base excision repair, mismatch repair, and nucleotide excision repair. They have since been joined by many scientists elucidating diverse aspects of these complex mechanisms that now constitute a thriving research field with many applications, notably for understanding oncogenesis and devising more effective therapies.

  9. DNA repair in reduced genome: the Mycoplasma model.

    PubMed

    Carvalho, Fabíola Marques; Fonseca, Marbella Maria; Batistuzzo De Medeiros, Sílvia; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella

    2005-11-01

    The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species. PMID:16153783

  10. Hemangioma excision (image)

    MedlinePlus

    A hemangioma is a non-cancerous (benign) growth of blood vessels. They are the most common benign blood vessel ( ... time and occasionally with medication. Large or disfiguring hemangiomas may require surgical excision.

  11. The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

    PubMed

    Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

    2014-07-01

    To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

  12. The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once

    PubMed Central

    Lafrance-Vanasse, Julien

    2014-01-01

    To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by Watson-Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases

  13. Methods to study transcription-coupled repair in chromatin.

    PubMed

    Gaillard, Hélène; Wellinger, Ralf Erik; Aguilera, Andrés

    2015-01-01

    The effect of endogenous and exogenous DNA damage on the cellular metabolism can be studied at the genetic and molecular level. A paradigmatic case is the repair of UV-induced pyrimidine dimers (PDs) by nucleotide excision repair (NER) in Saccharomyces cerevisiae. To follow the formation and repair of PDs at specific chromosome loci, cells are irradiated with UV-light and incubated in the dark to allow repair by NER. Upon DNA isolation, cyclobutane pyrimidine dimers, which account for about 90 % of PDs, can be cleaved in vitro by the DNA nicking activity of the T4 endonuclease V repair enzyme. Subsequently, strand-specific repair in a suitable restriction fragment is determined by denaturing gel electrophoresis followed by Southern blot and indirect end-labeling using a single-stranded DNA probe. Noteworthy, this protocol could potentially be adapted to other kind of DNA lesions, as long as a DNA nick is formed or a lesion-specific endonuclease is available.Transcription-coupled repair (TC-NER) is a sub-pathway of NER that catalyzes the repair of the transcribed strand of active genes. RNA polymerase II is essential for TC-NER, and its occupancy on a damaged template can be analyzed by chromatin immunoprecipitation (ChIP). In this chapter, we provide an up-dated protocol for both the DNA repair analysis and ChIP approaches to study TC-NER in yeast chromatin. PMID:25827885

  14. DNA repair meets the RNA world.

    PubMed

    Lee, Chow H

    2014-02-01

    The ability to repair damaged DNA and to maintain genome stability is the utmost importance for the survival of any species. Hence, it is not surprising to find that DNA repair mechanisms are evolutionarily conserved and are expected to evolve to maintain the existence of species. In the last few years, there has been an exponential increase in the evidence linking RNA processing with DNA repair programs. For instance, the well-studied DNA base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 can cleave RNA molecules, regulate mRNA levels, and associate physically with proteins involved in RNA processing. It is now clear that not only the expression of noncoding RNAs are changed upon DNA damage, they can modulate the expression of genes involved in the genome stability programs. The five reviews in this Forum provide the up-to-date knowledge on DNA repair, with a focus on BER, and a perspective on how the two ancient biochemical pathways are linked. The contributions demonstrate the complexity of such interactions, but also pointed out the opportunities for new therapeutic interventions. Future in vivo studies on the link between DNA repair processes and RNA metabolism should contribute to our basic understanding of physiology, disease, and treatment strategies.

  15. Inducible error-prone repair in B. subtilis. Final report, September 1, 1979-June 30, 1981

    SciTech Connect

    Yasbin, R. E.

    1981-06-01

    The research performed under this contract has been concentrated on the relationship between inducible DNA repair systems, mutagenesis and the competent state in the gram positive bacterium Bacillus subtilis. The following results have been obtained from this research: (1) competent Bacillus subtilis cells have been developed into a sensitive tester system for carcinogens; (2) competent B. subtilis cells have an efficient excision-repair system, however, this system will not function on bacteriophage DNA taken into the cell via the process of transfection; (3) DNA polymerase III is essential in the mechanism of the process of W-reactivation; (4) B. subtilis strains cured of their defective prophages have been isolated and are now being developed for gene cloning systems; (5) protoplasts of B. subtilis have been shown capable of acquiring DNA repair enzymes (i.e., enzyme therapy); and (6) a plasmid was characterized which enhanced inducible error-prone repair in a gram positive organism.

  16. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes.

    PubMed Central

    Demple, B; Herman, T; Chen, D S

    1991-01-01

    Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles of these enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that includes two bacterial AP endonucleases (ExoA protein of Streptococcus pneumoniae and exonuclease III of Escherichia coli) and Rrp1 protein of Drosophila melanogaster. Purified Ape protein lacks the 3'-exonuclease activity against undamaged DNA that is found in the bacterial and Drosophila enzymes, but the lack of obvious amino acid changes to account for this difference suggests that the various enzyme functions evolved by fine tuning a conserved active site. Expression of the active human enzyme in AP endonuclease-deficient E. coli conferred significant resistance to killing by the DNA-alkylating agent methyl methanesulfonate. The APE cDNA provides a molecular tool for analyzing the role of this central enzyme in maintaining genetic stability in humans. Images PMID:1722334

  17. Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

    PubMed Central

    Teo, I; Sedgwick, B; Demple, B; Li, B; Lindahl, T

    1984-01-01

    The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:6092060

  18. Submandibular gland excision.

    PubMed

    Crabtree, G M; Yarington, C T

    1988-10-01

    A review of 107 submandibular gland excisions was made to determine the relative frequency of neoplastic versus non-neoplastic disease. The results show a preponderance of non-neoplastic pathology. Benign tumors were found in 6% of glands, primary glandular and metastatic carcinoma in 3%. Timely treatment of a submandibular gland mass is indicated due to the relatively aggressive behavior of malignancies originating in this location.

  19. Laparoscopic Total Mesorectum Excision

    PubMed Central

    Quilici, F.A.; Cordeiro, F.; Reis, J.A.; Kagohara, O.; Simões Neto, J.

    2002-01-01

    The main controversy of colon-rectal laparoscopic surgery comes from its use as a cancer treatment. Two points deserve special attention: the incidence of portsite tumor implantation and the possibility of performing radical cancer surgery, such as total mesorectum excision. Once these points are addressed, the laparoscopic approach will be used routinely to treat rectal cancer. To clarify these points, 32 patients with cancer of the lower rectum participated in a special protocol that included preoperative radiotherapy and laparoscopic total mesorectum excision. All data were recorded. At the same time, all data recorded from the experience of a multicenter laparoscopic group (Brazilian Colorectal Laparoscopic Surgeons – 130 patients with tumor of the lower rectum) were analyzed and compared with the data provided by our patients. Analysis of the results suggests that a laparoscopic approach allows the same effective resection as that of conventional surgery and that preoperative irradiation does not influence the incidence of intraoperative complications. The extent of lymph nodal excision is similar to that obtained with open surgery, with an average of 12.3 lymph nodes dissected per specimen. The rate of local recurrence was 3.12%. No port site implantation of tumor was noted in this series of patients with cancer of the lower rectum. PMID:12113422

  20. Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing*

    PubMed Central

    Wang, Hongyan; Wang, Xiang; Chen, Guangnan; Zhang, Xiangming; Tang, Xiaobing; Park, Dongkyoo; Cucinotta, Francis A.; Yu, David S.; Deng, Xingming; Dynan, William S.; Doetsch, Paul W.; Wang, Ya

    2014-01-01

    High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy. PMID:25210033

  1. Effect of apelin on mitosis, apoptosis and DNA repair enzyme OGG 1/2 expression in intestinal cell lines IEC-6 and Caco-2.

    PubMed

    Antushevich, Hanna; Krawczynska, Agata; Kapica, Malgorzata; Herman, Andrzej Przemyslaw; Zabielski, Romuald

    2014-01-01

    Apelin is a regulatory peptide, identified as an endogenous ligand of the Apelin receptor (APJ). Both the apelin and the APJ were detected in brain, lung, heart, mammary gland, kidney, placenta, adipose tissues and the gastrointestinal tract. Apelin is considered an important regulatory gut peptide with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behaviour. The aim of the present study was to assess the effect of the apelin on mitosis, apoptosis and the expression of DNA repair enzyme (OGG 1/2), and APJ receptor in intestinal cell lines: rat crypt (IEC-6) and human enterocyte model (Caco-2). The cell cultures were incubated with the apelin-12 (10-8 M) for 4, 6, 12, 24 and 48 h and the apoptosis (caspase 3), mitosis (Ki-67) and DNA repair enzyme (OGG1/2) markers were studied by Real-Time qRT-PCR and immunofluorescent methods. The results of Real-Time qRT-PCR and immunocytochemical analysis showed that the levels of mRNAs were inversely related to the expression level of corresponding proteins. Immunofluorescent studies revealed inhibitory effect of apelin-12 on apoptosis, mitosis and the expression of OGG1/2 in the intestinal crypt cell line IEC-6. However, in the enterocyte model Caco-2 cells apelin stimulated apoptosis and mitosis, and reduced OGG1/2 expression. These findings suggest that apelin may be involved in the control of epithelial cell turnover in the gastrointestinal tract.

  2. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  3. More Than a Repair Enzyme: Aspergillus nidulans Photolyase-like CryA Is a Regulator of Sexual Development

    PubMed Central

    Bayram, Özgür; Biesemann, Christoph; Krappmann, Sven; Galland, Paul

    2008-01-01

    Cryptochromes are blue-light receptors that have presumably evolved from the DNA photolyase protein family, and the genomes of many organisms contain genes for both types of molecules. Both protein structures resemble each other, which suggests that light control and light protection share a common ancient origin. In the genome of the filamentous fungus Aspergillus nidulans, however, only one cryptochrome/photolyase-encoding gene, termed cryA, was identified. Deletion of the cryA gene triggers sexual differentiation under inappropriate culture conditions and results in up-regulation of transcripts encoding regulators of fruiting body formation. CryA is a protein whose N- and C-terminal synthetic green fluorescent protein fusions localize to the nucleus. CryA represses sexual development under UVA350-370 nm light both on plates and in submerged culture. Strikingly, CryA exhibits photorepair activity as demonstrated by heterologous complementation of a DNA repair-deficient Escherichia coli strain as well as overexpression in an A. nidulans uvsBΔ genetic background. This is in contrast to the single deletion cryAΔ strain, which does not show increased sensitivity toward UV-induced damage. In A. nidulans, cryA encodes a novel type of cryptochrome/photolyase that exhibits a regulatory function during light-dependent development and DNA repair activity. This represents a paradigm for the evolutionary transition between photolyases and cryptochromes. PMID:18495868

  4. PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities.

    PubMed

    Wei, Wei; Zhang, Chi; Liu, Ai-Lin; Xie, Shao-Hua; Chen, Xue-Min; Lu, Wen-Qing

    2009-09-10

    Both of benzo(a)pyrene (BaP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50microM) or/and PCB126 (0.01, 0.1, 1 and 10nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.

  5. Human MMH (OGG1) type 1a protein is a major enzyme for repair of 8-hydroxyguanine lesions in human cells.

    PubMed

    Monden, Y; Arai, T; Asano, M; Ohtsuka, E; Aburatani, H; Nishimura, S

    1999-05-19

    8-Hydroxyguanine (8-OH-G) is the site of a frequent mutagenic lesion of DNA, produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions. Recently, cDNA clones of human OGG1 homologues (hMMH) of four isoforms (type 1a, type 1b, type 1c, and type 2) were isolated. However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells. Here using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we show the expression of hMMH type 1a protein in many types of human cells and show that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity. Furthermore, we show that upon depletion of hMMH type 1a protein in a whole cell extract by its antibody, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesions in human cells. PMID:10329432

  6. Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers*

    PubMed Central

    Chen, Haobin; Giri, Nitai Charan; Zhang, Ronghe; Yamane, Kenichi; Zhang, Yi; Maroney, Michael; Costa, Max

    2010-01-01

    Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC50 [Ni(II)]) are 25 μm for JMJD1A and 7.5 μm for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC50 [Ni(II)] of 2.5 μm, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development. PMID:20042601

  7. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

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

    SciTech Connect

    Prakash, L.

    1992-04-01

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

  9. Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo.

    PubMed

    Lord, Tessa; Aitken, R John

    2015-10-01

    Oxidative DNA damage harbored by both spermatozoa and oocytes at the time of fertilization must be repaired prior to S-phase of the first mitotic division to reduce the risk of transversion mutations occurring in the zygote and subverting the normal patterns of cell differentiation and development. Of the characterised oxidative DNA lesions, 8-hydroxy-2'-deoxyguanosine (8OHdG) is particularly mutagenic. The current study reveals for the first time a marked acceleration of 8OHdG repair in the mouse oocyte/zygote by the base excision repair (BER) pathway following fertilization. Specifically, fertilization initiates post-translational modification to BER enzymes such as OGG1 and XRCC1, causing nuclear localisation and accelerated 8OHdG excision. Additionally, both the nuclear and mitochondrial genomes appear to benefit from increased protection against further 8OHdG formation by a fertilization-associated increase in glutathione peroxidase activity. The major limitation of the characterised 8OHdG repair system is the relatively low level of OGG1 expression in the oocyte, in contrast to the male germ line where it is the only constituent of the BER pathway. The male and female germ lines therefore collaborate in the repair of oxidative DNA damage, and oocytes are vulnerable to high levels of 8OHdG being carried into the zygote by the fertilizing spermatozoon. PMID:26234752

  10. 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.

  11. A Two-tiered compensatory response to loss of DNA repair modulates aging and stress response pathways

    PubMed Central

    Fensgård, Øyvind; Kassahun, Henok; Bombik, Izabela; Rognes, Torbjørn; Lindvall, Jessica Margareta; Nilsen, Hilde

    2010-01-01

    Activation of oxidative stress-responses and downregulation of insulin-like signaling (ILS) is seen in Nucleotide Excision Repair (NER) deficient segmental progeroid mice. Evidence suggests that this is a survival response to persistent transcription-blocking DNA damage, although the relevant lesions have not been identified. Here we show that loss of NTH-1, the only Base Excision Repair (BER) enzyme known to initiate repair of oxidative DNA damage inC. elegans, restores normal lifespan of the short-lived NER deficient xpa-1 mutant. Loss of NTH-1 leads to oxidative stress and global expression profile changes that involve upregulation of genes responding to endogenous stress and downregulation of ILS. A similar, but more extensive, transcriptomic shift is observed in the xpa-1 mutant whereas loss of both NTH-1 and XPA-1 elicits a different profile with downregulation of Aurora-B and Polo-like kinase 1 signaling networks as well as DNA repair and DNA damage response genes. The restoration of normal lifespan and absence oxidative stress responses in nth-1;xpa-1 indicate that BER contributes to generate transcription blocking lesions from oxidative DNA damage. Hence, our data strongly suggests that the DNA lesions relevant for aging are repair intermediates resulting from aberrant or attempted processing by BER of lesions normally repaired by NER. PMID:20382984

  12. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    SciTech Connect

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit; Choudhuri, Tathagata; Kundu, Chanakya Nath

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  13. Serum angiotensin-converting enzyme 2 is an independent risk factor for in-hospital mortality following open surgical repair of ruptured abdominal aortic aneurysm

    PubMed Central

    Nie, Wanpin; Wang, Yan; Yao, Kai; Wang, Zheng; Wu, Hao

    2016-01-01

    Open surgical repair (OSR) is a conventional surgical method used in the repair a ruptured abdominal aortic aneurysm (AAA); however, OSR results in high perioperative mortality rates. The level of serum angiotensin-converting enzyme 2 (ACE2) has been reported to be an independent risk factor for postoperative in-hospital mortality following major cardiopulmonary surgery. In the present study, the association of serum ACE2 levels with postoperative in-hospital mortality was investigated in patients undergoing OSR for ruptured AAA. The study enrolled 84 consecutive patients underwent OSR for ruptured AAA and were subsequently treated in the intensive care unit. Patients who succumbed postoperatively during hospitalization were defined as non-survivors. Serum ACE2 levels were measured in all patients prior to and following the surgery using ELISA kits. The results indicated that non-survivors showed significantly lower mean preoperative and postoperative serum ACE2 levels when compared with those in survivors. Multivariate logistic regression analysis also showed that, subsequent to adjusting for potential confounders, the serum ACE2 level on preoperative day 1 showed a significant negative association with the postoperative in-hospital mortality. This was confirmed by multivariate hazard ratio analysis, which showed that, subsequent to adjusting for the various potential confounders, the risk of postoperative in-hospital mortality remained significantly higher in the two lowest serum ACE2 level quartiles compared with that in the highest quartile on preoperative day 1. In conclusion, the present study provided the first evidence supporting that the serum ACE2 level is an independent risk factor for the in-hospital mortality following OSR for ruptured AAA. Furthermore, low serum ACE2 levels on preoperative day 1 were found to be associated with increased postoperative in-hospital mortality. Therefore, the serum ACE2 level on preoperative day 1 may be a potential

  14. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

    PubMed

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-01-01

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy. PMID:27589807

  15. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

    PubMed Central

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-01-01

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy. PMID:27589807

  16. Optimality in DNA repair.

    PubMed

    Richard, Morgiane; Fryett, Matthew; Miller, Samantha; Booth, Ian; Grebogi, Celso; Moura, Alessandro

    2012-01-01

    DNA within cells is subject to damage from various sources. Organisms have evolved a number of mechanisms to repair DNA damage. The activity of repair enzymes carries its own risk, however, because the repair of two nearby lesions may lead to the breakup of DNA and result in cell death. We propose a mathematical theory of the damage and repair process in the important scenario where lesions are caused in bursts. We use this model to show that there is an optimum level of repair enzymes within cells which optimises the cell's response to damage. This optimal level is explained as the best trade-off between fast repair and a low probability of causing double-stranded breaks. We derive our results analytically and test them using stochastic simulations, and compare our predictions with current biological knowledge. PMID:21945337

  17. Laparoscopic excision of subdiaphragmatic epidermoid cyst: a case report.

    PubMed

    Hagr, A; Laberge, J M; Nguyen, L T; Emil, S; Bernard, C; Patenaude, Y

    2001-06-01

    Retroperitoneal epidermoid cysts are rare. The authors report a case of an 11-year-old boy with an asymptomatic subdiaphragmatic cyst, which was found incidentally during an investigation for hypertension. At laparoscopy, the cyst was densely adherent to the diaphragm, resulting in a pneumothorax during dissection. Nevertheless, the excision and the diaphragmatic repair could be completed laparoscopically without complication. Microscopic examination showed an epidermoid cyst. No similar case has been reported in the literature.

  18. Hydrolytic function of Exo1 in mammalian mismatch repair

    PubMed Central

    Shao, Hongbing; Baitinger, Celia; Soderblom, Erik J.; Burdett, Vickers; Modrich, Paul

    2014-01-01

    Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function. PMID:24829455

  19. ppGpp couples transcription to DNA repair in E. coli.

    PubMed

    Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley; Proshkin, Sergey; Mironov, Alexander; Cashel, Michael; Nudler, Evgeny

    2016-05-20

    The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

  20. The DNA-mismatch repair enzyme hMSH2 modulates UV-B-induced cell cycle arrest and apoptosis in melanoma cells.

    PubMed

    Seifert, Markus; Scherer, Stefan J; Edelmann, Wilfried; Böhm, Markus; Meineke, Viktor; Löbrich, Markus; Tilgen, Wolfgang; Reichrath, Jörg

    2008-01-01

    The mechanisms by which the post-replicative DNA mismatch repair (MMR) enzyme MSH2 is involved in the complex response mechanisms to UV damage are yet to be clarified. Here, we show increased levels of MSH2 mRNA in malignant melanoma, metastases of melanoma, and melanoma cell (MeWo) lines as compared with melanocytic nevi or primary cultured benign melanocytes. UV-B treatment modulated MSH2 expression and silencing of MSH2 gene expression using small interfering RNA technology regulated UV-B-induced cell cycle arrest and apoptosis in human MeWo. We show that MSH2-deficient non-malignant mouse fibroblasts (MEF-/-) are partially resistant against UV-B-induced apoptosis and show reduced S-Phase accumulation. In addition, we show that an Msh2 point mutation (MEFGA) that affects MMR does not affect UV-B-induced apoptosis. In conclusion, we demonstrate that MSH2 modulates in human melanocytes both UV-B-induced cell cycle regulation and apoptosis, most likely via independent, uncoupled mechanisms.

  1. HAG3, a Histone Acetyltransferase, Affects UV-B Responses by Negatively Regulating the Expression of DNA Repair Enzymes and Sunscreen Content in Arabidopsis thaliana.

    PubMed

    Fina, Julieta P; Casati, Paula

    2015-07-01

    Histone acetylation is regulated by histone acetyltransferases and deacetylases. In Arabidopsis, there are 12 histone acetyltransferases and 18 deacetylases. Histone acetyltransferases are organized in four families: the GNAT/HAG, the MYST, the p300/CBP and the TAFII250 families. Previously, we demonstrated that Arabidopsis mutants in the two members of the MYST acetyltransferase family show increased DNA damage after UV-B irradiation. To investigate further the role of other histone acetyltransferases in UV-B responses, a putative role for enzymes of the GNAT family, HAG1, HAG2 and HAG3, was analyzed. HAG transcripts are not UV-B regulated; however, hag3 RNA interference (RNAi) transgenic plants show a lower inhibition of leaf and root growth by UV-B, higher levels of UV-B-absorbing compounds and less UV-B-induced DNA damage than Wassilewskija (Ws) plants, while hag1 RNAi transgenic plants and hag2 mutants do not show significant differences from wild-type plants. Transcripts for UV-B-regulated genes are highly expressed under control conditions in the absence of UV-B in hag3 RNAi transgenic plants, suggesting that the higher UV-B tolerance may be due to increased levels of proteins that participate in UV-B responses. Together, our data provide evidence that HAG3, directly or indirectly, participates in UV-B-induced DNA damage repair and signaling.

  2. Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

    PubMed

    Barzilay, G; Walker, L J; Robson, C N; Hickson, I D

    1995-05-11

    HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein. PMID:7784208

  3. DNA repair of a single UV photoproduct in a designed nucleosome

    SciTech Connect

    Kosmoskil, Joseph V.; Ackerman, Eric J. ); Smerdon, Michael J.

    2001-08-28

    Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is > 50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA ( 165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

  4. Defective repair of 8-hydroxyguanine in mitochondria of MCF-7 and MDA-MB-468 human breast cancer cell lines.

    PubMed

    Mambo, Elizabeth; Nyaga, Simon G; Bohr, Vilhelm A; Evans, Michele K

    2002-03-01

    Breast cancer is one of the major causes of mortality among women in the United States. Although the causes of breast cancer remain unclear, it has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Recently, defective oxidative DNA damage repair has been implicated in breast tumorigenesis. The major oxidative DNA lesion, 8-hydroxyguanine (8-oxoG), is increased in breast cancer, suggesting that this lesion may play a crucial role in the etiology of breast cancer. However, it is not known whether the repair of 8-oxoG or other oxidative base lesions is altered during breast carcinogenesis. We examined the ability of nuclear and mitochondrial extracts of two human breast cancer cell lines, MCF-7 and MDA-MB-468, to repair 8-oxoG lesion. We report that mitochondrial extracts from the two breast cancer cell lines are defective in the base excision repair of 8-oxoG relative to two noncancer cell lines. We also show that the incision activity of 8-oxoG was significantly lower in mitochondrial than in nuclear extracts in the breast cancer cell lines. The defective mitochondrial repair activity was not attributable to lower levels of human 8-hydroxyguanine DNA glycosylase, the base excision repair enzyme known to incise 8-oxoG in DNA. The repair of thymine glycol, another major oxidative DNA base lesion that blocks transcription and causes cell death, was similar in cancer and noncancer cells. Furthermore, nuclear extracts incised thymine glycol with a much higher efficiency than 8-oxoG. These data provide evidence for defective repair of 8-oxoG in mitochondria of MCF-7 and MDA-MB-468 breast cancer cell lines. These results may implicate 8-oxoG repair mechanisms in mitochondria of certain breast cancers.

  5. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

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

  6. Curcumin prevents DNA damage and enhances the repair potential in a chronically arsenic-exposed human population in West Bengal, India.

    PubMed

    Roy, Madhumita; Sinha, Dona; Mukherjee, Sutapa; Biswas, Jaydip

    2011-03-01

    Induction of oxidative stress and inhibition of DNA repair are possible modes of arsenic-induced carcinogenesis. In West Bengal, India, several districts contain high levels of arsenic, which are far above the WHO-recommended standard. Prevention of arsenic-induced oxidative stress and induction of repair enzymes by curcumin, an active ingredient of turmeric, may be an effective strategy to combat the adverse effects of arsenic. This study aimed at observing the role of curcumin in reducing 8-hydroxy-20-deoxyguanosine formation and enhancing DNA repair capacity in the arsenic-exposed population of West Bengal. Chronically arsenic-exposed volunteers (n= 66), who were asymptomatic, were selected for this study. Our results indicated that curcumin suppressed the 8-hydroxy-20-deoxyguanosine level and OGG1 expression, which were increased by arsenic. Curcumin also induced DNA repair enzymes involved in both base excision repair and nonhomologous end-joining pathways. In this study, both the protein expression and genetic profile were observed for poly-ADP-ribose polymerase 1, DNA b polymerase, X ray repair cross complement 1, DNA ligase III, DNA protein kinase catalytic sub-unit, X ray repair cross-complement 4, DNA ligase IV, and topoisomerase II b. The results indicated that arsenic-inhibited DNA repair was induced by curcumin, both at protein and genetic levels. Thus, curcumin intervention may be a useful modality for the prevention of arsenic-induced carcinogenesis. PMID:21332098

  7. Curcumin prevents DNA damage and enhances the repair potential in a chronically arsenic-exposed human population in West Bengal, India.

    PubMed

    Roy, Madhumita; Sinha, Dona; Mukherjee, Sutapa; Biswas, Jaydip

    2011-03-01

    Induction of oxidative stress and inhibition of DNA repair are possible modes of arsenic-induced carcinogenesis. In West Bengal, India, several districts contain high levels of arsenic, which are far above the WHO-recommended standard. Prevention of arsenic-induced oxidative stress and induction of repair enzymes by curcumin, an active ingredient of turmeric, may be an effective strategy to combat the adverse effects of arsenic. This study aimed at observing the role of curcumin in reducing 8-hydroxy-20-deoxyguanosine formation and enhancing DNA repair capacity in the arsenic-exposed population of West Bengal. Chronically arsenic-exposed volunteers (n= 66), who were asymptomatic, were selected for this study. Our results indicated that curcumin suppressed the 8-hydroxy-20-deoxyguanosine level and OGG1 expression, which were increased by arsenic. Curcumin also induced DNA repair enzymes involved in both base excision repair and nonhomologous end-joining pathways. In this study, both the protein expression and genetic profile were observed for poly-ADP-ribose polymerase 1, DNA b polymerase, X ray repair cross complement 1, DNA ligase III, DNA protein kinase catalytic sub-unit, X ray repair cross-complement 4, DNA ligase IV, and topoisomerase II b. The results indicated that arsenic-inhibited DNA repair was induced by curcumin, both at protein and genetic levels. Thus, curcumin intervention may be a useful modality for the prevention of arsenic-induced carcinogenesis.

  8. Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

    PubMed Central

    Liu, Geyi; Aronovich, Elena L.; Cui, Zongbin; Whitley, Chester B.; Hackett, Perry B.

    2007-01-01

    A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase–transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. PMID:15133768

  9. Preventive Long-Term Effects of a Topical Film-Forming Medical Device with Ultra-High UV Protection Filters and DNA Repair Enzyme in Xeroderma Pigmentosum: A Retrospective Study of Eight Cases

    PubMed Central

    Giustini, Sandra; Miraglia, Emanuele; Berardesca, Enzo; Milani, Massimo; Calvieri, Stefano

    2014-01-01

    Skin cancer is common in xeroderma pigmentosum (XP) due to a DNA repair mechanisms genetic defect. Ultraviolet (UV) exposure is the main cause of increased incidence of actinic keratosis (AK), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) observed in XP subjects. Photoprotection is therefore a mandatory strategy in order to reduce skin damage. A topical DNA repair enzyme has been shown to slow down the development of skin lesions in XP. However, there are no data regarding the effects of photoprotection combined with DNA repair strategies in this clinical setting. A film-forming medical device containing the DNA repair enzyme photolyase and very high-protection UV filters (Eryfotona AK-NMSC, Ery) is currently available. We report retrospective data regarding the use of Ery in 8 patients (5 women, 3 men) with a diagnosis of XP treated for at least 12 consecutive months, comparing the rate of new skin lesions (AK, BCC and SCC) during active treatment with Ery and during 12 months just before the use of the product. New AK, BCC and SCC mean lesion numbers during the 1-year Ery treatment were 5, 3 and 0, respectively in comparison with 14, 6.8 and 3 lesions, respectively during the 1-year pre-treatment period. Ery use was associated with a 65% reduction in appearance of new AK lesions and with 56 and 100% reductions in the incidence of new BCC and SCC lesions, respectively. These data suggest that topical use of photoprotection and DNA repair enzyme could help lower skin cancer lesions in XP. Control prospective trials are advisable in this clinical setting. PMID:25408650

  10. CdSe/ZnS Quantum Dots trigger DNA repair and antioxidant enzyme systems in Medicago sativa cells in suspension culture

    PubMed Central

    2013-01-01

    Background Nanoparticles appear to be promising devices for application in the agriculture and food industries, but information regarding the response of plants to contact with nano-devices is scarce. Toxic effects may be imposed depending on the type and concentration of nanoparticle as well as time of exposure. A number of mechanisms may underlie the ability of nanoparticles to cause genotoxicity, besides the activation of ROS scavenging mechanisms. In a previous study, we showed that plant cells accumulate 3-Mercaptopropanoic acid-CdSe/ZnS quantum dots (MPA-CdSe/ZnS QD) in their cytosol and nucleus and increased production of ROS in a dose dependent manner when exposed to QD and that a concentration of 10 nM should be cyto-compatible. Results When Medicago sativa cells were exposed to 10, 50 and 100 nM MPA-CdSe/ZnS QD a correspondent increase in the activity of Superoxide dismutase, Catalase and Glutathione reductase was registered. Different versions of the COMET assay were used to assess the genotoxicity of MPA-CdSe/ZnS QD. The number of DNA single and double strand breaks increased with increasing concentrations of MPA-CdSe/ZnS QD. At the highest concentrations, tested purine bases were more oxidized than the pyrimidine ones. The transcription of the DNA repair enzymes Formamidopyrimidine DNA glycosylase, Tyrosyl-DNA phosphodiesterase I and DNA Topoisomerase I was up-regulated in the presence of increasing concentrations of MPA-CdSe/ZnS QD. Conclusions Concentrations as low as 10 nM MPA-CdSe/ZnS Quantum Dots are cytotoxic and genotoxic to plant cells, although not lethal. This sets a limit for the concentrations to be used when practical applications using nanodevices of this type on plants are being considered. This work describes for the first time the genotoxic effect of Quantum Dots in plant cells and demonstrates that both the DNA repair genes (Tdp1β, Top1β and Fpg) and the ROS scavenging mechanisms are activated when MPA-CdSe/ZnS QD contact M. sativa

  11. Endogenous DNA Damage and Repair Enzymes: -A short summary of the scientific achievements of Tomas Lindahl, Nobel Laureate in Chemistry 2015.

    PubMed

    Klungland, Arne; Yang, Yun-Gui

    2016-06-01

    Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career. PMID:26689322

  12. Endogenous DNA Damage and Repair Enzymes: -A short summary of the scientific achievements of Tomas Lindahl, Nobel Laureate in Chemistry 2015.

    PubMed

    Klungland, Arne; Yang, Yun-Gui

    2016-06-01

    Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career.

  13. The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylase.

    PubMed

    Le Meur, Rémy; Culard, Françoise; Nadan, Virginie; Goffinont, Stéphane; Coste, Franck; Guerin, Martine; Loth, Karine; Landon, Céline; Castaing, Bertrand

    2015-10-01

    The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.

  14. Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice

    PubMed Central

    Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M.; Gorodnya, Olena M.; Ruchko, Mykhaylo V.; Potter, Barry J.; Wilson, Glenn L.; Gillespie, Mark N.

    2013-01-01

    This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH2O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH2O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH2O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury. PMID:23241530

  15. Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

    PubMed

    Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M; Gorodnya, Olena M; Ruchko, Mykhaylo V; Potter, Barry J; Wilson, Glenn L; Gillespie, Mark N; Parker, James C

    2013-02-15

    This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

  16. An Unprecedented Nucleic Acid Capture Mechanism for Excision of DNA Damage

    PubMed Central

    Rubinson, Emily H.; Gowda, A.S. Prakasha; Spratt, Thomas E.; Gold, Barry; Eichman, Brandt F.

    2014-01-01

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA targeting anticancer compounds. The basis for glycosylase specificity toward N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here, we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched, and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing the first illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how AlkD’s HEAT-repeats distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation. PMID:20927102

  17. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    SciTech Connect

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F.

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  18. 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.

  19. New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

    PubMed

    Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

    2015-05-01

    Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory

  20. Protein oxidation, UVA and human DNA repair.

    PubMed

    Karran, Peter; Brem, Reto

    2016-08-01

    Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency - with particular reference to NER and skin cancer risk.

  1. Repair of ultraviolet-light-induced damage

    SciTech Connect

    Sutherland, B.M.

    1981-01-01

    Studies are reviewed which present three major new findings in the photobiology of skin. First, detectable numbers of dimers are formed even at sub-erythymal doses. Second, excision of dimers is much more rapid than would be predicted from results obtained in cell culture. Third, comparison of the rates of excision and photoreactivation in skin indicates that in normal sunlight exposure, photoreactivation may well be the predominant repair pathway in skin. (ACR)

  2. Molecular Regulation of UV-Induced DNA Repair

    PubMed Central

    Shah, Palak; He, Yu-Ying

    2014-01-01

    Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the U.S., as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors. PMID:25534312

  3. Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

    PubMed Central

    Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

    2016-01-01

    Background Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Methods Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. Results No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. Conclusion BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which

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

    SciTech Connect

    Quirk, W.A.

    1993-04-01

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

  5. Recognition and excision properties of 8-halogenated-7-deaza-2'-deoxyguanosine as 8-oxo-2'-deoxyguanosine analogues and Fpg and hOGG1 inhibitors.

    PubMed

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2015-05-26

    Cellular DNA continuously suffers various types of damage, and unrepaired damage increases disease progression risk. 8-Oxo-2'-deoxyguanine (8-oxo-dG) is excised by repair enzymes, and their analogues are of interest as inhibitors and as bioprobes for study of these enzymes. We have developed 8-halogenated-7-deaza-2'-deoxyguanosine derivatives that resemble 8-oxo-dG in that they adopt the syn conformation. In this study, we investigated their effects on Fpg (formamidopyrimidine DNA glycosylase) and hOGG1 (human 8-oxoguanine DNA N-glycosylase 1). Relative to 8-oxo-dG, Cl- and Br-deaza-dG were good substrates for Fpg, whereas they were less efficient substrates for hOGG1. Kinetics and binding experiments indicated that, although hOGG1 effectively binds Cl- and Br-deaza-dG analogues with low Km values, their lower kcat values result in low glycosylase activities. The benefits of the high binding affinities and low reactivities of 8-oxo-dG analogues with hOGG1 have been successfully applied to the competitive inhibition of the excision of 8-oxoguanine from duplex DNA by hOGG1.

  6. Structural basis of HIV-1 resistance to AZT by excision.

    PubMed

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D; Clark, Arthur D; Hou, Xiaorong; Frenkel, Yulia V; Gaffney, Barbara L; Jones, Roger A; Boyer, Paul L; Hughes, Stephen H; Sarafianos, Stefan G; Arnold, Eddy

    2010-10-01

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  7. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  8. Hypomorphic PCNA mutation underlies a human DNA repair disorder

    PubMed Central

    Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

    2014-01-01

    Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

  9. Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, June 1, 1979-May 31, 1980

    SciTech Connect

    Billen, D.; Hadden, C.T.

    1980-01-01

    Progress is reported in the following research areas: (1) DNA repair in Bacillus subtilis; (2) postreplication repair in Rec/sup -/ mutants of B. subtilis; (3) photobiology of halogenated DNA; (4) effects of caffeine on pyrimidine dimer excision and postreplication repair in B. subtilis; and (5) DNA repair in toluene-treated Escherichia coli. (ACR)

  10. Use of enzymatic assay to evaluate UV-induced DNA repair in human and embryonic chick fibroblasts and multinucleate heterokaryons derived from both.

    PubMed

    Paterson, M C; Lohman, P H

    1975-01-01

    nuclei from both strains. Experimental conditions were selected so that UV-induced damage resided only in DNA foreign to the repair enzymes under study. Our results strongly suggest that repair enzyme(s) coded for by either fusion partner can remove dimer-containing sites from the DNA of the other with an efficiency comparable to that attained when acting on its own DNA in unfused, parental cells. Further, the light-requiring repair process supplied by the chick is more proficient at operating on these sites in human DNA than is excision-repair, the parallel mechanism available to human cells for this purpose. PMID:1238081

  11. Mfd as a central partner of transcription coupled repair.

    PubMed

    Monnet, Jordan; Grange, Wilfried; Strick, Terence R; Joly, Nicolas

    2013-01-01

    Transcription-coupled repair (TCR) is one of the key of the nucleotide excision repair (NER) pathways required to preserve genome integrity. Although understanding TCR is still a major challenge, recent single-molecule experiments have brought new insights into the initial steps of TCR leading to new perspectives.

  12. Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

    PubMed Central

    Marteijn, Jurgen A.; Bekker-Jensen, Simon; Mailand, Niels; Lans, Hannes; Schwertman, Petra; Gourdin, Audrey M.; Dantuma, Nico P.; Lukas, Jiri

    2009-01-01

    Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification. PMID:19797077

  13. Human DNA repair genes.

    PubMed

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

    2001-02-16

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

  14. Specific Inhibition of NEIL-initiated repair of oxidized base damage in human genome by copper and iron: potential etiological linkage to neurodegenerative diseases.

    PubMed

    Hegde, Muralidhar L; Hegde, Pavana M; Holthauzen, Luis M F; Hazra, Tapas K; Rao, K S Jagannatha; Mitra, Sankar

    2010-09-10

    Dyshomeostasis of transition metals iron and copper as well as accumulation of oxidative DNA damage have been implicated in multitude of human neurodegenerative diseases, including Alzheimer disease and Parkinson disease. These metals oxidize DNA bases by generating reactive oxygen species. Most oxidized bases in mammalian genomes are repaired via the base excision repair pathway, initiated with one of four major DNA glycosylases: NTH1 or OGG1 (of the Nth family) or NEIL1 or NEIL2 (of the Nei family). Here we show that Fe(II/III) and Cu(II) at physiological levels bind to NEIL1 and NEIL2 to alter their secondary structure and strongly inhibit repair of mutagenic 5-hydroxyuracil, a common cytosine oxidation product, both in vitro and in neuroblastoma (SH-SY5Y) cell extract by affecting the base excision and AP lyase activities of NEILs. The specificity of iron/copper inhibition of NEILs is indicated by a lack of similar inhibition of OGG1, which also indicated that the inhibition is due to metal binding to the enzymes and not DNA. Fluorescence and surface plasmon resonance studies show submicromolar binding of copper/iron to NEILs but not OGG1. Furthermore, Fe(II) inhibits the interaction of NEIL1 with downstream base excision repair proteins DNA polymerase beta and flap endonuclease-1 by 4-6-fold. These results indicate that iron/copper overload in the neurodegenerative diseases could act as a double-edged sword by both increasing oxidative genome damage and preventing their repair. Interestingly, specific chelators, including the natural chemopreventive compound curcumin, reverse the inhibition of NEILs both in vitro and in cells, suggesting their therapeutic potential. PMID:20622253

  15. Epigenetic self-regulation of developmental excision of an internal eliminated sequence on Paramecium tetraurelia.

    PubMed

    Duharcourt, S; Butler, A; Meyer, E

    1995-08-15

    Differentiation of the somatic macronucleus of ciliates after sexual events involves the programmed excision of thousands of single-copy internal eliminated sequences (IESs) from the germ-line genome. We have studied two cell lines of Paramecium tetraurelia that have identical germ-line genomes but differ in their macronuclear genomes. In the IES- cell line, a 222-bp IES interrupting a coding sequence is reproducibly excised during macronuclear differentiation, whereas it is not in the IES+ cell line. In a cross between the two lines, the developmental alternative in maternally inherited, suggesting that it is epigenetically controlled by the old (prezygotic) macronucleus in each cell. Transformation of the macronucleus of both lines with plasmids carrying fragments of either version of the gene shows that the presence of the IES sequence in the old macronucleus results in retention of the IES in the new macronuclear genome of sexual progeny. This could be attributable to (1) inhibition of excision, or (2) repair of a double-strand gap left in the genomic sequence after constitutive excision of the IES, by a polymerization mechanism using a homologous IES+ template from the old macronucleus. The latter possibility is ruled out by experiments showing that modified IESs can inhibit excision without being copied in the new macronuclear genome. Possible mechanisms are discussed in the light of a quantitative analysis of excision inhibition by the maternal IES sequence. PMID:7649484

  16. State cigarette excise taxes - United States, 2009.

    PubMed

    2010-04-01

    Increasing the price of cigarettes can reduce smoking substantially by discouraging initiation among youths and young adults, prompting quit attempts, and reducing average cigarette consumption among those who continue to smoke. Increasing cigarette excise taxes is one of the most effective tobacco control policies because it directly increases cigarette prices, thereby reducing cigarette use and smoking-related death and disease. All states and the District of Columbia (DC) impose an excise tax on cigarettes. Because many states increased their cigarette excise taxes in 2009, CDC conducted a survey of these tax increases. For this report, CDC reviewed data contained in a legislative database to identify cigarette excise tax legislation that was enacted during 2009 by the 50 states and DC. During that period, 15 states (including DC), increased their state excise tax on cigarettes, increasing the national mean from $1.18 per pack in 2008 to $1.34 per pack in 2009. However, none of the 15 states dedicated any of the new excise tax revenue by statute to tobacco control. Additionally, for the first time, two states (Connecticut and Rhode Island) had excise tax rates of at least $3.00 per pack. Additional increases in cigarette excise taxes, and dedication of all resulting revenues to tobacco control and prevention programs at levels recommended by CDC, could result in further reductions in smoking and associated morbidity and mortality.

  17. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  18. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2012-04-01 2011-04-01 true Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  19. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2014-04-01 2014-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  20. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  1. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2013-04-01 2013-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  2. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2011-04-01 2011-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  3. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  4. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  5. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  6. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  7. Stellate excision of malignancies on the auricles.

    PubMed

    Bumsted, R M; Ceilley, R I

    1980-01-01

    Primary excision of malignancies on the auricles with the standard "wedge technique" may produce lateral protrusion of the helix. A stellate-type excision eliminates the possibility of this undesirable effect and allows the surgeon to close large defects in the helix. The technique is described in detail.

  8. Clubfoot repair

    MedlinePlus

    ... release; Talipes equinovarus - repair; Tibialis anterior tendon transfer Images Clubfoot repair - series References Kelly DM. Congenital Anomalies ... provided herein should not be used during any medical emergency or for the diagnosis or treatment of ...

  9. Tendon repair

    MedlinePlus

    Repair of tendon ... Tendon repair can be performed using: Local anesthesia (the immediate area of the surgery is pain-free) ... a cut on the skin over the injured tendon. The damaged or torn ends of the tendon ...

  10. Colocutaneous Fistula after Open Inguinal Hernia Repair

    PubMed Central

    Kallis, Panayiotis; Koronakis, Nikolaos; Hadjicostas, Panayiotis

    2016-01-01

    The plug-and-patch technique is frequently used for the open repair of inguinal hernias; however, serious complications may arise on rare occasions. We present the case of a 69-year-old patient who presented with a colocutaneous fistula with the sigmoid colon 9 years after the repair of a left sliding inguinal hernia with the plug-and-patch technique. The patient underwent sigmoidectomy and excision of the fistulous track. He was discharged on postoperative day 5 and had an uneventful recovery. Although such complications are reported rarely, the surgeon must be aware of them when deciding upon the method of hernia repair. PMID:27738544

  11. Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining.

    PubMed

    Maezawa, So; Nakano, Saori; Kuniya, Takaaki; Koiwai, Osamu; Koiwai, Kotaro

    2016-01-01

    We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one- or two-base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells. PMID:27047738

  12. Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle.

    PubMed

    Madura, K; Prakash, S; Prakash, L

    1990-02-25

    The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation. Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation. RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV. RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs. However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth. These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination. The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

  13. DNA Damage and Repair in Eukaryotic Cells

    PubMed Central

    Painter, R. B.

    1974-01-01

    Damage in DNA after irradiation can be classified into five kinds: base damage, single-strand breaks, double-strand breaks, DNA–DNA cross-linking, and DNA-protein cross-linking. Of these, repair of base damage is the best understood. In eukaryotes, at least three repair systems are known that can deal with base damage: photoreactivation, excision repair, and post-replication repair. Photoreactivation is specific for UV-induced damage and occurs widely throughout the biosphere, although it seems to be absent from placental mammals. Excision repair is present in prokaryotes and in animals but does not seem to be present in plants. Post-replication repair is poorly understood. Recent reports indicate that growing points in mammalian DNA simply skip past UV-induced lesions, leaving gaps in newly made DNA that are subsequently filled in by de novo synthesis. Evidence that this concept is oversimplified or incorrect is presented.—Single-strand breaks are induced by ionizing radiation but most cells can rapidly repair most or all of them, even after supralethal doses. The chemistry of the fragments formed when breaks are induced by ionizing radiation is complex and poorly understood. Therefore, the intermediate steps in the repair of single-strand breaks are unknown. Double-strand breaks and the two kinds of cross-linking have been studied very little and almost nothing is known about their mechanisms for repair.—The role of mammalian DNA repair in mutations is not known. Although there is evidence that defective repair can lead to cancer and/or premature aging in humans, the relationship between the molecular defects and the diseased state remains obscure. PMID:4442699

  14. Histological assessment of tangentially excised burn eschars

    PubMed Central

    Gurfinkel, Reuven; Rosenberg, Lior; Cohen, Sarit; Cohen, Arnon; Barezovsky, Alex; Cagnano, Emanuela; Singer, Adam J

    2010-01-01

    BACKGROUND: The burn eschar serves as a medium for bacterial growth and a source of local and systemic infection. To prevent or minimize these complications, it is important to debride the eschar as early as possible. OBJECTIVE: To identify the presence of viable skin within the excisions by examining tangentially excised burn eschars. METHODS: A total of 146 samples of burned human tissue were removed during 54 routine sharp tangential excision procedures (using dermatomes). The samples were histologically examined to identify the relative thickness of the dead, intermediate and viable layers. RESULTS: The mean (± SD) thickness of the excised samples was 1.7±1.1 mm. The sacrificed viable tissue (mean thickness 0.7±0.8 mm) occupied 41.2% of the entire thickness of the excision. In 32 biopsies (21.8%; 95% CI 16.0 to 29.3), the excision did not reach viable skin. Only eight biopsies (5.4%; 95% CI 2.8 to 10.1) contained all of the necrotic tissue without removing viable tissue. CONCLUSIONS: The thickness of a single tangentially excised layer of eschar is not much greater than the actual thickness of the entire skin and often contains viable tissue. Because surgical debridement is insufficiently selective, more selective means of debriding burn eschars should be explored. PMID:21886431

  15. RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli.

    PubMed

    Nagel, R; Chan, A

    1999-03-10

    Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating. PMID:10102036

  16. Whence flavins? Redox-active ribonucleotides link metabolism and genome repair to the RNA world.

    PubMed

    Nguyen, Khiem Van; Burrows, Cynthia J

    2012-12-18

    Present-day organisms are under constant environmental stress that damages bases in DNA, leading to mutations. Without DNA repair processes to correct these errors, such damage would be catastrophic. Organisms in all kingdoms have repair processes ranging from direct reversal to base excision and nucleotide excision repair, and the recently characterized giant viruses also include these mechanisms. At what point in the evolution of genomes did active repair mechanisms become critical? In particular, how did early RNA genomes protect themselves from UV photodamage that would have hampered nonenzymatic replication and led to a mutation rate too high to pass on accurate sequence information from one generation to the next? Photolyase is a widespread and phylogenetically ancient enzyme that utilizes longer wavelength light to cleave thymine dimers in DNA produced via photodamage. The protein serves as a binding scaffold but does not contribute to the catalytic chemistry; the action of the dinucleotide cofactor FADH(2) breaks the chemical bonds. This small bit of RNA, hailed as a "fossil of the RNA World," contains the flavin heterocycle, whose redox activity has been harnessed for myriad functions of life from metabolism to DNA repair. In present-day biochemistry, flavin biosynthesis begins with guanosine and proceeds through seven steps catalyzed by protein-based enzymes. This leads to the question of how flavins originally evolved. Did the RNA world include ancestral RNA bases with greater redox activity than G, A, C, and U that were capable of photorepair of uracil dimers? Could those ancestral bases have chemically evolved to the current flavin structure? Or did flavins already exist from prebiotic chemical synthesis? And were they then co-opted as catalysts for repair sometime after metabolism was established? In this Account, we analyze simple derivatives of guanosine and other bases that show two prerequisites for flavin-like photolyase activity: a significantly

  17. DNA repair in Mycoplasma gallisepticum

    PubMed Central

    2013-01-01

    Background DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a “minimal cell” concept. Results In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. Conclusions Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase. PMID:24148612

  18. Small RNAs Recruit Chromatin-Modifying Enzymes MMSET and Tip60 to Reconfigure Damaged DNA upon Double-Strand Break and Facilitate Repair.

    PubMed

    Wang, Qinhong; Goldstein, Michael

    2016-04-01

    Recent reports have demonstrated that DNA double-strand break (DSB)-induced small RNAs (diRNA) play an important role in the DNA damage response (DDR). However, the molecular mechanism by which diRNAs regulate the DDR remains unclear. Here, we report that Dicer- and Drosha-dependent diRNAs function as guiding molecules to promote the recruitment of the methyltransferase MMSET (WHSC1) and the acetyltransferase Tip60 (KAT5) to the DSB, where local levels of histone H4 di- and tri-methylation at lysine 20 (H4K20me2, 3) and H4 acetylation at lysine 16 (H4K16Ac) were enhanced. These histone modification events resulted in an open, flexible chromatin configuration, as indicated by the increased release of histones γH2AX, H2AX, and H3 from damaged chromatin. Furthermore, we found that diRNA-associated AGO2 interacted with MMSET and Tip60 and that the diRNA binding and catalytic activities of AGO2 were dispensable for the interaction but required for the recruitment of MMSET and Tip60 to DSBs. Consequently, diRNA-mediated chromatin remodeling promoted DSB repair by enhancing the recruitment of Rad51 and BRCA1 to the DSB site. Taken together, our findings reveal an unexpected direct role for diRNAs in regulating chromatin remodeling to facilitate DSB repair, revealing a new layer of DDR regulation involving specialized RNA molecules. Cancer Res; 76(7); 1904-15. ©2016 AACR. PMID:26822153

  19. Simple and easy reconstruction of nail matrix lesion using lateral finger flap after excision of digital mucous cyst.

    PubMed

    Okochi, Masayuki; Saito, Masami; Ueda, Kazuki

    2016-01-01

    We treated nine patients with skin defect produced by digital mucous cyst (DMC) excision on the finger and toe using lateral finger flap (LFF). The postoperative scars were esthetically acceptable and no recurrence of mucous cysts was observed. Our LFF is a simple method to repair minor distal dorsal finger defects. PMID:27583263

  20. Mutagen sensitivity of Drosophila melanogaster. VII Alkylation mutagenesis of mature and immature oocytes of the excision-deficient mus (2) 201/sup D1/ mutant

    SciTech Connect

    Badaruddin, A.S.; Dusenbery, R.L.; Smith, P.D.

    1984-01-01

    The mus (2) 201/sup D1/ strain has normal meiotic processes but is absolutely defective in excision repair ability following alkylation treatment. The authors examined what effect total excision deficiency has on induced mutagenesis in these stages of oogenesis. Excision-proficient cn/sup 35/ females and excision-deficient cn/sup 35/ mus (2) 201/sup D1/ females were exposed to MMS in phosphate buffer, and subsequently mated to Basc males for five broods. From their data they conclude: first, even at this low exposure to MMS, the mus (2) 201/sup D1/ females exhibit high infertility in broods 3 through 5, indicative of extensive cell killing in the excision-deficient genotype; second, for the excision-proficient cn/sup 35/ strain, the frequency of induced mutagenesis in brood 1 is approximately threefold that observed in brood 2; third, by comparing the induced frequencies for broods 1 and 2 between two strains, the data demonstrate a very substantial increase in mutagenesis in the excision-deficient strain over that observed for the control strain. The data clearly demonstrate the extensive contribution that the excision repair process makes during oogenesis and early embryogenesis in protecting the female and zygotic genomes from mutational consequences arising fron DNA damage.

  1. Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals.

    PubMed

    Sidorkina, O; Dizdaroglu, M; Laval, J

    2001-09-15

    The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA

  2. DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

    PubMed

    López-Camarillo, César; Lopez-Casamichana, Mavil; Weber, Christian; Guillen, Nancy; Orozco, Esther; Marchat, Laurence A

    2009-12-01

    Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

  3. Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions

    PubMed Central

    Tsaalbi-Shtylik, Anastasia; Ferrás, Cristina; Pauw, Bea; Hendriks, Giel; Temviriyanukul, Piya; Carlée, Leone; Calléja, Fabienne; van Hees, Sandrine; Akagi, Jun-Ichi; Iwai, Shigenori; Hanaoka, Fumio; Jansen, Jacob G.

    2015-01-01

    In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa–Atr–Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects. PMID:25869665

  4. Giant rhinophyma: Excision with coblation assisted surgery.

    PubMed

    Sahin, Caner; Turker, Mesut; Celasun, Bulent

    2014-01-01

    An 83-year-old man presented with an unusually severe case of rhinophyma. Giant rhinopyhma is very rare in literature. The giant lesion was widely excised using sharp surgical incision and coblation assisted surgery. Using direct coblation to the nasal dorsum may cause edema in the surrounding tissue. There was minimal edema in surrounding tissue using this technique. A full thickness-skin graft was applied after excision. Cosmetic and functional postoperative results were satisfactory.

  5. Giant rhinophyma: Excision with coblation assisted surgery

    PubMed Central

    Sahin, Caner; Turker, Mesut; Celasun, Bulent

    2014-01-01

    An 83-year-old man presented with an unusually severe case of rhinophyma. Giant rhinopyhma is very rare in literature. The giant lesion was widely excised using sharp surgical incision and coblation assisted surgery. Using direct coblation to the nasal dorsum may cause edema in the surrounding tissue. There was minimal edema in surrounding tissue using this technique. A full thickness-skin graft was applied after excision. Cosmetic and functional postoperative results were satisfactory. PMID:25593440

  6. Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

    PubMed

    Fukuyo, Masaki; Nakano, Toshiaki; Zhang, Yingbiao; Furuta, Yoshikazu; Ishikawa, Ken; Watanabe-Matsui, Miki; Yano, Hirokazu; Hamakawa, Takeshi; Ide, Hiroshi; Kobayashi, Ichizo

    2015-03-11

    The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

  7. Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    PubMed Central

    Fukuyo, Masaki; Nakano, Toshiaki; Zhang, Yingbiao; Furuta, Yoshikazu; Ishikawa, Ken; Watanabe-Matsui, Miki; Yano, Hirokazu; Hamakawa, Takeshi; Ide, Hiroshi; Kobayashi, Ichizo

    2015-01-01

    The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes. PMID:25697504

  8. An experimental double-blind irradiation study of a novel topical product (TPF 50) compared to other topical products with DNA repair enzymes, antioxidants, and growth factors with sunscreens: implications for preventing skin aging and cancer.

    PubMed

    Emanuele, Enzo; Spencer, James M; Braun, Martin

    2014-03-01

    The exposure to ultraviolet radiation (UVR) is a major risk factor for skin aging and the development of non-melanoma skin cancer (NMSC). Although traditional sunscreens remain the mainstay for the prevention of UVR-induced skin damage, they cannot ensure a complete protection against the whole spectrum of molecular lesions associated with UVR exposure. The formation of helix-distorting photoproducts such as cyclobutane pyrimidine dimers (CPD), as well as oxidative damage to DNA bases, including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8OHdG) are among the key DNA lesions associated with photoaging and tumorigenesis. Besides DNA lesions, UVR-induced formation of free radicals can result in protein carbonylation (PC), a major form of irreversible protein damage that inactivates their biological function. This study compares a complex novel topical product (TPF50) consisting of three actives, ie, 1) traditional physical sunscreens (SPF 50), 2) a liposome-encapsulated DNA repair enzymes complex (photolyase, endonuclease, and 8-oxoguanine glycosylase [OGG1]), and 3) a potent antioxidant complex (carnosine, arazine, ergothionine) to existing products. Specifically, we assessed the ability of TFP50 vs those of DNA repair and antioxidant and growth factor topical products used with SPF 50 sunscreens in preventing CPD, 8OHdG, and PC formation in human skin biopsies after experimental irradiations. In head-to-head comparison studies, TPF50 showed the best efficacy in reducing all of the three molecular markers. The results indicated that the three TPF50 components had a synergistic effect in reducing CPD and PC, but not 8OHdG. Taken together, our results indicate that TPF50 improves the genomic and proteomic integrity of skin cells after repeated exposure to UVR, ultimately reducing the risk of skin aging and NMSC.

  9. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex.

    PubMed

    Bouvet, Mickaël; Imbert, Isabelle; Subissi, Lorenzo; Gluais, Laure; Canard, Bruno; Decroly, Etienne

    2012-06-12

    The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1-16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3' to 5' direction as well as a single mismatched nucleotide at the 3'-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3'-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2'-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism. PMID:22635272

  10. 29 CFR 794.121 - Exclusion of excise taxes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Exclusion of excise taxes. 794.121 Section 794.121 Labor... Exclusion of excise taxes. The computation of the annual gross volume of sales of the enterprise for purposes of section 7(b)(3) is made “exclusive of excise taxes.” It will be noted that the excise...

  11. DNA repair systems as targets of cadmium toxicity

    SciTech Connect

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios . E-mail: theocharis@ath.forthnet.gr

    2006-06-15

    Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity.

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

    PubMed Central

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

    2013-01-01

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

  13. SNPs of GSTM1, T1, P1, epoxide hydrolase and DNA repair enzyme XRCC1 and risk of urinary transitional cell carcinoma in southwestern Taiwan

    SciTech Connect

    Hsu, L.-I; Chiu, Allen W.; Huan, Steven K.; Chen, C.-L.; Wang, Y.-H.; Hsieh, F.-I; Chou, W.-L.; Wang, L.-H.; Chen, C.-J.

    2008-04-15

    A hospital-based case-control study was conducted near a former black-foot disease (BFD)-endemic area in southwestern Taiwan to examine the possible risk factors and genetic susceptibility for urinary transitional cell carcinoma (TCC). A total of 221 patients with pathologically confirmed TCC and 223 age-sex-matched control subjects from urology outpatient clinics were recruited between 1998 and 2002. The results showed that residency in the BFD area and consumption of well water for more than 10 years was a strong factor on urinary cancer risk (odds ratio [OR],8.16, 95% confidence interval [CI],3.34-19.90, p < 0.0001). Dose response relationship between average arsenic concentration in well water and TCC risk was also observed. Cigarette smoking played a relatively minor role in urinary carcinogenesis in this study. The GSTP1 Ile105Val A {yields} G polymorphism was significantly associated with cancer risk (A/G + G/G: OR = 0.60, 95%CI = 0.39-0.94, p = 0.02), and the effect of Val105 allele was largely confined to the subjects diagnosed earlier than 55 years old (A/G + G/G: OR,0.29; 95% CI, 0.09-0.87, p = 0.03). The results suggest that GSTP1 is a candidate for susceptibility locus and Ile105 allele may predispose individuals to early-onset urinary TCC. The GSTM1 null genotype was associated with tumors of high-invasiveness (OR,2.21; 95% CI, 1.34-4.73) as well as with early-onset TCC risk (OR,2.53; 95% CI, 0.97-6.59). Our preliminary results showed the XRCC1 Arg194Trp were associated with arsenic-related urinary TCC and the interaction between the genotype and the exposure was statistically significant. The modulating effect of the GSTM1, GSTT1, GSTP1 Ile105Val, EPHX Tyr113His and XRCC1 Arg280His on arsenic-related TCC risk was also suggestive. These observations implied that impaired metabolism of carcinogenic exposure as well as impaired DNA repair function play an important role in arsenic-related urinary transitional cell carcinogenesis.

  14. Ultraviolet damage, DNA repair and vitamin D in nonmelanoma skin cancer and in malignant melanoma: an update.

    PubMed

    Reichrath, Jörg; Rass, Knuth

    2014-01-01

    Skin exposure with UV radiation (UV) is the main cause of skin cancer development. Epidemiological data indicate that excessive or cumulative UV exposure takes place years and decades before the resulting malignancies arise. The most important defense mechanisms that protect human skin against UV radiation involve melanin synthesis and active repair mechanisms. DNA is the major target of direct or indirect UV-induced cellular damage. Low pigmentation capacity in white Caucasians and rare congenital defects in DNA repair are mainly responsible for protection failures. The important function of nucleotide excision DNA repair (NER) to protect against skin cancer becomes obvious by the rare genetic disease xeroderma pigmentosum, in which diverse NER genes are mutated. In animal models, it has been demonstrated that UVB is more effective to induce skin cancer than UVA. UV-induced DNA photoproducts are able to cause specific mutations (UV-signature) in susceptible genes for squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). In SCC development, UV-signature mutations in the p53 tumor suppressor gene are the most common event, as precancerous lesions reveal -80% and SCCs > 90% UV-specific p53 mutations. Mutations in Hedgehog pathway related genes, especially PTCH1, are well known to represent the most significant pathogenic event in BCC. However, specific UV-induced mutations can be found only in -50% of sporadic BCCs. Thus, cumulative UVB radiation cannot be considered to represent the only etiologic risk factor for BCC development. During the last decades, experimental animal models, including genetically engineered mice, the Xiphophorus hybrid fish, the South American oppossum and human skin xenografts, have further elucidated the important role of the DNA repair system in the multi-step process of UV-induced melanomagenesis. An increasing body of evidence now indicates that nucleotide excision repair is not the only DNA repair pathway that is involved in UV

  15. Complications of Mohs' chemosurgical excision of eyelid and canthal tumors.

    PubMed