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Effects of oxidative DNA damage on transcription by RNA polymerases
DNA Damage and Repair 5.9
INVESTIGATION OF DNA BASE DAMAGE BY ENZYMATICALLY-GENERATED OXYGEN RADICALS IN CULTURED FETAL LUNG CELLS James F. Belliveau, Donna Corvese and Alan B. Weitberg Roger Willilams Cancer Center, Brown University and Providence College, Providence, RI 02908, U.S.A. We investigated the formation of modified DNA bases caused by exposure of intact cells to enzymatically generated oxygen radicals at biologically relevant levels. The possible presence or enhancement of five modified bases was measured in aqueous DNA and intact human cell samples (MRC-5 fetal lung cells) exposed to oxygen radicals (hypoxanthine/xanthine oxidase) by GC/MS analysis. In control samples of both aqueous DNA and intact cells, the modified base, 5-hydroxyuracil, was observed at low levels (approximately one per six thousand thymines). At oxygen radical levels that produce genetic lesions in intact MRC-5 cells, the 5-hydroxyuracil level was increased by an order of magnitude and 8-hydroxyguanine was present (which was not observed in the control sample). [The 5-hydroxyuracil level also increased by a factor of 4 in treated DNA samples.] 5-Hydroxycytosine, 7, 8 cis-thymine glycol and 8-hydroxyadenine were not observed in either the treated or control samples. These data indicate that DNA base modificacations may be a major factor at the molecular level for the genetic lesions caused by exposure of intact cells to oxygen radicals.
5.11
FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE OF E.COLI EXCISES IMIDAZOLE RING-OPENED PURINES FROM DNA EXPOSED TO IONIZING RADIATION Serge Boi%eux I, Ewa Gajews~i 2, Miral ~izdaroglu ~ and Jacques Laval ~ UA 158 CNRS, U 140 INSERM, Institut ~ustave Roussy, 94800 Villejuif, FRANCE NIST, Gaithersburg, Maryland 20899. The formamidopyrimidine DNA glycosylase (FPG protein) of ~.coli is a 30 kDa protein which is endowed with two enzymatic activities; a DNA glycosylase activity which removes imidazole ring-opened N7 Methylguanine residues, and a lyase activity which incises DNA at apurinic sites. Formamidopyrimidine residues derived from guanine and adenine are generated in DNA exposed to ionizing radiations and oxidizing agents. The spectrum of DNA base modifications induced by radiation was analysed by gas chromatography-mass spectrometry (GCMS). Incubation of ~ i r r a d i a t e d DNA with homogeneous FPG protein results in the liberation of Fapy-Adenine and FapyGuanine residues as determined by GC-MS analysis of the products of the reaction. Neither 8-OH-Guanine, 8-OHAdenine or modified pyrimidines including thymine and cytosine glycols are excised at a significant rate. These results show that the FPG protein is one of the enzymes responsible for the repair of free radicals induced damages in DNA. Therefore, FPG protein contributes to cellular processes that counteract the deleterious effects of reactive oxygen species.
SEX AS A RESPONSE TO OXIDATIVE DNA DAMAGE Carol Bcrnstein and Virginia Johns Microbiology and Immunology, University of Arizona, Tucson, AZ 85724
47 5.10
Sexual reproduction is very widespread. Among higher plants, only 8% of species lack the two key aspects of sexual reproduction: outcrossing and meiosis. Among animals, less than 0.1% of species are parthenogenic. Thus, most species have sex. What is sex for? We have proposed that one major advantage of sex is its promotion of recombinational repair of DNA damage during meiosis. During meiosis, homologous chromosomes are brought into close juxtaposition. At this time recombinational repair takes place. If repair is a major function of the sexual cycle, this should be testable. H909 is a byproduct of oxidative cellular metabolism arid ffas been proposed to be an important natural cause of DNA damage in cells. Treatment of the asexual form of a facultatively sexual/asexual eucaryote with H909 may cause it to enter the sexual cycle more freq~en-tly. Therefore, we treated vegetative cells of Schizosaccharomyces pombe with H202 to see if sexual reproduction increases. Among untre~te~ stationary phase S. pombe populations, the sexual spores produced by meiosis represent about 1% of the ceils. However, treatment of late-exponential-phasevegetative cells with HgO 2 increased the percentage of meiotic spores in stationary phase by 4- to 18-fold. This suggests that sex evolved, in part, as a response to DNA damage.
EFFECTS OF OXIDATIVE DNA DAMAGE ON TRANSCRIPTION BY RNA POLYMERASES Suzanne Byrd, Daniel Reines, and Paul W. Doetsch Dept. of Biochemistry, Emory University, Atlanta, GA 30322 Transcriptional regulation is a key element in the control of gene expression. Deficiencies in the various steps of transcription (i.e., initiation, elongation, or termination) of an essential gene may have catastrophic consequences for a cell. In terminally differentiated cells, the removal of RNA polymerase-blocking lesions could be viewed as a critical function for DNA repair systems in such cells. Oxidative damage to DNA has been implicated in the degenerative changes associated with a number of age-related pathologies. Thymine glycol is a major oxidative DNA base damage product that can be produced spontaneously in normal cells or by certain chemicals and ionizing radiation. Thymine glycol is not a strong premutagenic lesion but it efficiently blocks DNA polymerase. This lesion as well as other oxidatively damaged bases are recognized and removed in eukaryotic cells by the DNA repair enzyme redoxyendonuclease which we have recently identified in bovine neuronal tissue; however, very little information exists on the direct effects of such oxidative base damage products on the transcriptional machinery. A 3'-extended DNA segment encoding a region of the human H3.3 histone gene was utilized to determine the effects of oxidative DNA base damage on the ability of pure E. colicore RNA polymerase to transcribe such a template. Thymine glycol was chemically introduced into this template prior to in vitro transcription. Analysis of the transcripts by high resolution gel electrophoresis indicates that oxidative DNA base damage products may inhibit transcription directly by causing arrest or pausing of RNA polymerase at or near the site of damage. The effects of oxidative DNA damage on templates transcribed by mammalian RNA polymerase II in promoter-based transcription systems will also be discussed. These results support the notion that an important function of DNA repair systems in differentiated cells is to ensure efficient transcription of genes necessary for normal cellular function. Supported by NIH grants CA42607 and BRSG S07 RR05364.
5.12
INVESTIGATION OF DNA BASE DAMAGE BY ENZYMATICALLY-GENERATED OXYGEN RADICALS IN CULTURED FETAL LUNG CELLS James F. Belliveau, Donna Corvese and Alan B. Weitberg Roger Willilams Cancer Center, Brown University and Providence College, Providence, RI 02908, U.S.A. We investigated the formation of modified DNA bases caused by exposure of intact cells to enzymatically generated oxygen radicals at biologically relevant levels. The possible presence or enhancement of five modified bases was measured in aqueous DNA and intact human cell samples (MRC-5 fetal lung cells) exposed to oxygen radicals (hypoxanthine/xanthine oxidase) by GC/MS analysis. In control samples of both aqueous DNA and intact cells, the modified base, 5-hydroxyuracil, was observed at low levels (approximately one per six thousand thymines). At oxygen radical levels that produce genetic lesions in intact MRC-5 cells, the 5-hydroxyuracil level was increased by an order of magnitude and 8-hydroxyguanine was present (which was not observed in the control sample). [The 5-hydroxyuracil level also increased by a factor of 4 in treated DNA samples.] 5-Hydroxycytosine, 7, 8 cis-thymine glycol and 8-hydroxyadenine were not observed in either the treated or control samples. These data indicate that DNA base modificacations may be a major factor at the molecular level for the genetic lesions caused by exposure of intact cells to oxygen radicals.
5.11
FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE OF E.COLI EXCISES IMIDAZOLE RING-OPENED PURINES FROM DNA EXPOSED TO IONIZING RADIATION Serge Boi%eux I, Ewa Gajews~i 2, Miral ~izdaroglu ~ and Jacques Laval ~ UA 158 CNRS, U 140 INSERM, Institut ~ustave Roussy, 94800 Villejuif, FRANCE NIST, Gaithersburg, Maryland 20899. The formamidopyrimidine DNA glycosylase (FPG protein) of ~.coli is a 30 kDa protein which is endowed with two enzymatic activities; a DNA glycosylase activity which removes imidazole ring-opened N7 Methylguanine residues, and a lyase activity which incises DNA at apurinic sites. Formamidopyrimidine residues derived from guanine and adenine are generated in DNA exposed to ionizing radiations and oxidizing agents. The spectrum of DNA base modifications induced by radiation was analysed by gas chromatography-mass spectrometry (GCMS). Incubation of ~ i r r a d i a t e d DNA with homogeneous FPG protein results in the liberation of Fapy-Adenine and FapyGuanine residues as determined by GC-MS analysis of the products of the reaction. Neither 8-OH-Guanine, 8-OHAdenine or modified pyrimidines including thymine and cytosine glycols are excised at a significant rate. These results show that the FPG protein is one of the enzymes responsible for the repair of free radicals induced damages in DNA. Therefore, FPG protein contributes to cellular processes that counteract the deleterious effects of reactive oxygen species.
SEX AS A RESPONSE TO OXIDATIVE DNA DAMAGE Carol Bcrnstein and Virginia Johns Microbiology and Immunology, University of Arizona, Tucson, AZ 85724
47 5.10
Sexual reproduction is very widespread. Among higher plants, only 8% of species lack the two key aspects of sexual reproduction: outcrossing and meiosis. Among animals, less than 0.1% of species are parthenogenic. Thus, most species have sex. What is sex for? We have proposed that one major advantage of sex is its promotion of recombinational repair of DNA damage during meiosis. During meiosis, homologous chromosomes are brought into close juxtaposition. At this time recombinational repair takes place. If repair is a major function of the sexual cycle, this should be testable. H909 is a byproduct of oxidative cellular metabolism arid ffas been proposed to be an important natural cause of DNA damage in cells. Treatment of the asexual form of a facultatively sexual/asexual eucaryote with H909 may cause it to enter the sexual cycle more freq~en-tly. Therefore, we treated vegetative cells of Schizosaccharomyces pombe with H202 to see if sexual reproduction increases. Among untre~te~ stationary phase S. pombe populations, the sexual spores produced by meiosis represent about 1% of the ceils. However, treatment of late-exponential-phasevegetative cells with HgO 2 increased the percentage of meiotic spores in stationary phase by 4- to 18-fold. This suggests that sex evolved, in part, as a response to DNA damage.
EFFECTS OF OXIDATIVE DNA DAMAGE ON TRANSCRIPTION BY RNA POLYMERASES Suzanne Byrd, Daniel Reines, and Paul W. Doetsch Dept. of Biochemistry, Emory University, Atlanta, GA 30322 Transcriptional regulation is a key element in the control of gene expression. Deficiencies in the various steps of transcription (i.e., initiation, elongation, or termination) of an essential gene may have catastrophic consequences for a cell. In terminally differentiated cells, the removal of RNA polymerase-blocking lesions could be viewed as a critical function for DNA repair systems in such cells. Oxidative damage to DNA has been implicated in the degenerative changes associated with a number of age-related pathologies. Thymine glycol is a major oxidative DNA base damage product that can be produced spontaneously in normal cells or by certain chemicals and ionizing radiation. Thymine glycol is not a strong premutagenic lesion but it efficiently blocks DNA polymerase. This lesion as well as other oxidatively damaged bases are recognized and removed in eukaryotic cells by the DNA repair enzyme redoxyendonuclease which we have recently identified in bovine neuronal tissue; however, very little information exists on the direct effects of such oxidative base damage products on the transcriptional machinery. A 3'-extended DNA segment encoding a region of the human H3.3 histone gene was utilized to determine the effects of oxidative DNA base damage on the ability of pure E. colicore RNA polymerase to transcribe such a template. Thymine glycol was chemically introduced into this template prior to in vitro transcription. Analysis of the transcripts by high resolution gel electrophoresis indicates that oxidative DNA base damage products may inhibit transcription directly by causing arrest or pausing of RNA polymerase at or near the site of damage. The effects of oxidative DNA damage on templates transcribed by mammalian RNA polymerase II in promoter-based transcription systems will also be discussed. These results support the notion that an important function of DNA repair systems in differentiated cells is to ensure efficient transcription of genes necessary for normal cellular function. Supported by NIH grants CA42607 and BRSG S07 RR05364.
5.12