PL9800397
Institute of Biochemistry and Biophysics Polish Academy of Sciences
Abstracts of the Conference on:
MECHANISMS OF DNA REPAIR AND MUTAGENESIS
on the 100th Anniversary of the Discovery of Polonium and Radium
Warsaw, October 8-11, 1997 ISBN 83-906782-6-8
Wydawca: Instytut Biochemii i Biofizyki Polskiej Akademii Nauk 02-106 Warszawa ill. Pawiriskiego 5a
Warszawa 1997
Druk: AKCES Agencja Wydawnicza i Reklamowa tel.: 39-16-17
Cena: 6,90 zt Institute of Biochemistry and Biophysics
Polish Academy of Sciences
Abstracts of the Conference on:
MECHANISMS OF DNA REPAIR AND MUTAGENESIS
on the 100th Anniversary of The Discovery of Polonium and Radium
Warsaw, October 8-11, 1997 Organizing Committee of the Conference:
President: Barbara Tudek Vice-president: Irena Pietrzykowska Anna B^benek Zygmunt Ciesla Iwona Fijalkowska Maria-Anna Grajziewicz Elzbieta Grzesiuk Celina Janion Piotr Jonczyk Jarostaw Kusmierek JanilSZ Siedlecki (Institute of Oncology, Warsaw) Ewa &ledziewska-G6jska
Honorary Committee of the Conference:
Wtodzimierz Zagorski-Ostoja (Director of IBB PAS) David Shugar TadeUSZ Chojnacki (Secretary of Biological Sciences Dept, PAS) FikllS (Secretary of Committee of Biochemistry, PAS) Program
The Program of the Conference on:
MECHANISMS OF DNA REPAIR AND MUTAGENESIS on the 100th Anniversary of The Discovery of Polonium and Radium Warszawa, October 8-11, 1997
WEDNESDAY 8 OCTOBER 1997
930 j ooo OPENING CEREMONY
Session I: Oxidative DNA Damage and its Genetic Consequences Chairperson: B. Singer
1000- 103 L. Ernster, Dept. of Biochemistry, Aarhenius Lab., University of Stockholm, Sweden Jwo different aspects of oxidative DNA damage: mitochondrial disorders and cancer chemotherapy"
1035 - 1105 S. Boiteux, Commissariat a L'Energie Atomique, Centre d'Etudes de Fonteney-aux-Roses, Fonteney-aux-Roses, France „ Repair of 8-Oxoguanine in eukaryotic cells: the Oggl enzymes" j j io _ j |40 ^ Collins, Rowett Research Institute, Aberdeen, Great Britain »Oxidative DNA damage in human cells: antioxidant protection and DNA repair"
II45-1200 coffee break
1200 - 1230 R. Olinski, Dept. of Clinical Biochemistry, University of Medical Science, Bydgoszcz, Poland ..Oxidative DNA base modification as factors in carcinogenesis and AIDS" Program
1235 - 1255 B. Tudek, Institute of Biochemistry an Biophysics, PAS, Warsaw, Poland „ Formation of imidazole ring-opened purines in DNA and their biological consequences"
U^-U00 lunch
Chairperson: S. Boiteux
1405 - 1435 S. Loft, Dept. of Pharmacalogy, University of Copenhagen, Denmark „ Estimation ofoxidative DNA damage in man from urinary excretion of repair products"
14 -15 A. Barbin, unit of Enviromental Carcinogenesis, IARC, Lyon, France „ Formation of DNA etheno adducts in rodents and humans and their role in carcinogenesis"
15-15 I. Felzenszwalb, Universidade do Estado do Rio de Janeiro, Instituto de Biologia, Rio de Janeiro, Brazil „Hydrogen peroxide effects in Escherichia coli cells"
1550-1605 coffee break
Chairperson: J. Drake
1605 - 1635 B. Bridges, University of Sussex, Brighton, Great Britain
MOxidative DNA damage as a cause of spontaneous mutation in non-dividing cells" 1640 - 1710 R. C. von Borstel, University of Alberta, Edmonton, Canada „ Repair of radiation damage by DNA and deoxyribonucleosides"
1715 - 1745 D. Shugar, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland
18 30- departure to the welcome party
19oo_21oo WELCOME PARTY Program
THURSDAY 9 OCTOBER 1997
SESSION II DNA Repair in Prokaryotes and Mechanisms of Mutagenesis Chairperson: J. T. Kusmierek
9-9 B. Singer, Donner Laboratory - LBL, University of California, Berkeley, USA „ Structure-function relationships in replication and repair of carcinogen-damaged DNA"
935 - 1005 J. W. Drake, NIEHS, Research Triangle Park, USA „Autonomous polymerase fidelity"
1010 - 1040 K. Bebenek, NIEHS, Research Triangle Park, USA „Structure and functions of DNA polymerases"
1045 - II10 I. Fijalkowska, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland ..Unequal fidelity of leading and lagging strand replication on E.coli chromosome"
II15-II30 coffee break
Chairperson: H. Hayatsu
1130 - 1200 R. M. Schaaper, NIEHS, Research Triangle Park, USA „ Mutagenesis and DNA replication fidelity in Escherichia coil"
1205 - 1235 R. P. Fuchs, Groupe de Cancerogenese et de Mutagenese, Moleculaire et Structural, I.B.M.C du CNRS, Starsbourg, France „Steps during translesion synthesis and replication blocking lesions"
1240 - 1310 R. T. Johnson, University of Cambridge, Cambridge, United Kingdom „Solutions to replication on damaged DNA templates" Program
1315 -1415 lunch
Chairperson: T. Kunkel
1415 - 1445 C. Lawrence, University of Rochester, USA ,,DNA polymerase zeta and the control ofDNA damage induced mutagenesis in eukaryotes"
1450 - 15 R. Devoret, Laboratoire Mutagenese et Cancerogenese, Institute Curie, Orsay, France ,, The mechanism of SOS mutagenesis involves dynamic interaction between recombination and mutagenesis proteins"
1525 - 1555 R. Woodgate, NICHD, NIH, Bethesda, USA ^Structural insights into the regulation of SOS-mutagenesis"
16°°-1615 coffee break
Chairperson: Z. Ciesla
1615 - 1635 A. Bebenek, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland ,,A new gene in E.coli K12, isfA may be involved in down regulation of the SOS system" 1640 - 1700 I. Pietrzykowska, Institute of Biochemistry and Biophysics, PAS.Warsaw, Poland »Studies on UV-induced mutagenesis not related to DNA replication"
1705 - 1725 C. Janion, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland „ Effect ofTnlO on the survival and mutation frequency of halogen light-irradiated E.coli strain AB1157"
18'5 departure to the Theatre Program
MUDAY 10 OCTOBER 1997
900 - 1300 Warsaw sightseeing or free time
1300-1400 lunch
Chairperson: D. Shugar
14 -14 H. Hayatsu, Okayama University, Japan „ Mechanism of mutagenesis ofcytosine analogs"
1435 - 1505 Y. Pavlov, Dept. of Genetics and Breeding, Sankt-Petersburg State University, Sankt Petersburg, Russia ,,Mutagenic pathways of the base analog, 6-N-hydroxylaminopurine in bacteria and yeast"
15-15 J. T. KuSmierek, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland ,,Template-directed base pairing of 2-chlorodeoxyadenosine catalysed by AMV reverse transcriptase"
1535-1550 coffee break
Session III sponsored by POLISH NETWORK of MBCM UNESCO and POLISCH ACADEMY OF SCIENCE DNA Repair in Eukaryotes and its Relation to Human Diseases Chairperson: A. Collins
1550 - 16 ° M. Z. Zdzienicka, Dept. of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden, The Netherlands »Genetic basis of radiation sensitivity of mammalian cells"
1625 - 1655 T. Kunkel, NIEHS, Research Triangle Park, USA ,,Studies of eukaryotic DNA mismatch repair"
17-17 J. Jiricny, University of Zurich and The Paul Scherrer Institute, Switzerland ..Biochemistry of G/T mismatch repair in human cells" Program
1735 - 1805 B. Salles, IPBS, CNRS, Toulouse, France Mucleotide excision repair: in vitro analysis"
1810 - 1840 L. H. F. Mullenders, Dept. of Genetics, Radiation Genetics and Chemical Mutagenesis, Medical Genetics Centre, Netherlands „ Processing of structurally different lesions by nucieotide excision repair"
1900-2130 DINNER, Poster Session
SATURDAY 11 OCTOBER 1997 Chairperson: M. Zdzienicka
900 . 930 G. Russev, Institutte of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria „ Assessing NER activity in different genome regions"
935 - 1005 M. Pirsel, Dept. of Molecular Genetics, Cancer Research Institute, Bratislava, Slovakia ,,DNA repair domains"
1010 - 1040 G. P. Margison, Dept. of Carcinogenesis, Peterson Institute for Cancer Research, Manchester, Great Britain ,,DNA alkylation damage, repair and clinical significance"
1045 - II15 K. Kleibl, Cancer Research Institute, Bratislava, Slovakia Jnactivation of alkyltransferase to enhance chemoterapy"
II20-II35 coffee break
Chairperson: L. H. F. Mullenders
II35- 1205 T. R. O'Connor, National Medical Centre, City of Hope, USA „Repair of DNA alkylating agent damage in human cells"
1210 - 1240 F. Larminat, Laboratoire de Pharmacologie et de Toxicologie Foundamentales du CNRS, Toulouse, France ^Mammalian cellular responses to DNA cross-linking agents" Program
1245 - 1315 L. Daya-Grosjean, Institute sur le Cancer - IFC 1, Villejuif, France Jhe role ofp53 in DNA repair deficient syndromes hypersensitive to UV light"
1320-1430 lunch
Chairperson: R. C. von Borstel
1430 . 1500 A. Larsen, Institut Gustave Roussy, LA147 CNRS, Villejuif, France „ Influence of the oncogene suppressor protein p53 on the sensitivity of tumor cells to DNA topoisomerase inhibitors"
15-15 R. Hancock, Laval University Cancer Research Center, Quebeck, Canada ,,Oncogenic transformation resulting from dysfunction of the DNA cleavage-religation reaction of topoisomerase II"
15 -16 M. Nivard, Dept. of Radiation Genetics and Chemical Mutagenesis, Leiden University, Leiden, The Netherlands „ The impact of DNA repair on chemically induced germ cell mutagenesis"
16-16 R. Stetina, Lab. of Developmental Toxicology, Institute of Experimental Medicine, Olesnice v Orlickych Horach, Czech Republic ,,DNA damage in lymphocytes and aortal endothelia of diabetic and cholesterol-fed rats"
1650 - 1715 CLOSING LECTURES Session I: Oxidative DNA Damage and its Genetic Consequences
TWO DIFFERENT ASPECTS OF OXIDATIVE DNA DAMAGE: MITOCHONDRIAL DISORDERS AND CANCER CHEMOTHERAPY Lars Ernster Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden.
It is now well established that reactive oxygen species (ROS) can damage DNA, causing base oxidations, strand-breaks and cross-linking. Mitochondrial DNA is especially prone to oxidative damage, because of its steady exposure to ROS, its lack of histones, and of efficient repair mechanism. Many mitochondrial disorders, including a number of neurodegenerative diseases, as well as the normal process of aging and cell death, have their origin in oxidative damage to mitochondrial DNA. In addition, oxidative damage to mitochondrial DNA is a self-amplifying process, giving rise to increased ROS formation via the respiratory chain and thereby an increase in DNA damage, which explains the progressive nature of these diseases. Another aspect of oxidative DNA damage relates to the mode of action of various drugs used in cancer chemotherapy. In this case, various quinones and nitro compounds can be reductively activated by cellular enzymes, especially the two-electron transferring quinone reductase known as DT diaphorase, an enzyme which is overexpressed in many tumors, to give rise to compounds that can attack DNA by forming adducts or by causing strand-breaks or cross- linking, and thereby inhibit cell proliferation. The present lecture will briefly summerize current information concerning these two pathophysiological aspects of oxidative DNA damage, i.e., the oxidative damage of mitochondrial DNA, with emphasis on its implications for degenerative diseases, aging and cell death; and the application of drug- induced DNA damage to cancer chemotherapy. Session I: Oxidative DNA Damage and its Genetic Consequences
REPAIR OF 8-0X0GUANINE IN EUKARYOTES: THE OGGl ENZYMES Serge Boiteux and J. Pablo Rasicella CEA, Departement de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, F-92265-Fontenay aux Roses (France)
An oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8- OxoG), is a critical DNA lesion in term of mutagenesis and carcinogenesis. The OGGl gene of Saccharomyces cerevisiae codes for a 43-kDa protein and is mapped to chromosome XIII. The yOggl protein possesses both a DNA glycosylase activity that excises 8-OxoG and another activity that nicks DNA at abasic site. Studies of the catalytic mechanism demonstrate that the yOggl protein is a DNA glycosylase / AP lyase which proceeds via the formation of a transient covalent imino enzyme-DNA intermediate. The OGGl gene has been disrupted yielding the oggl::TRPl mutant. The oggl mutant of Saccharomyces cerevisiae is a mutator that specifically accumulates GC to TA transversions. Using the OGGl sequence, we were able to retrieve several human cDNA clones displaying close to 40% identity with the deduced yeast protein sequence. Northern blot analysis showed that the corresponding gene is expressed in all the human tissues analyzed. The cloned coding sequence was then expressed in Escherichia coli. Extracts from these cultures displayed a DNA glycosylase / AP lyase activity on DNA carrying an 8-OxoG/C base pair. Moreover, when hOGGl was expressed in a yeast oggl mutant strain, it was able to complement the mutator phenotype. The hOGGl gene was localized at 3p25, a locus which is frequently lost in lung cancer. We conclude that Oggl enzymes contribute to the maintenance of genetic stability in simple eukaryotes. Furthermore, we suggest that inactivation of hOGGl may predispose cells toward carcinogenesis. Session I: Oxidative DNA Damage and its Genetic Consequences
OXIDATIVE DAMAGE IN HUMAN CELLS: ANTIOXIDANT PROTECTION AND DNA REPAIR. Andrew Collins, Susan Duthic, Laurence Fillion, Aigun Ma, Mihalis Panayiotides and Mark Taylor Rowett Research Institute, Greenbum Road, Bucksburn, Aberdeen, AB21 9SB, UK
We have used the alkaline comet assay (single cell gel electrophoresis) to measure oxidative DNA damage - both strand breaks and oxidised bases. The latter are detected by incubating DNA with a lesion-specific endonuclease, either endonuclease III (for oxidised pyrimidines), or formamidopyrimidine glycosylase, which recognises altered purines including 8-oxo-guanine. We have investigated the hypothesis that antioxidants in the diet can protect against oxidative damage. At the end of a 20-week supplementation with vitamin C, vitamin E and p-carotene, oxidised pyrimidines were decreased in lymphocyte DNA of both smokers and non-smokers, compared with placebo groups. Lymphocytes from the supplemented groups were also more resistant to oxidative damages induced in vitro by H2O2. A single large dose of antioxidant is similarly effective at increasing resistance to H2O2 damage and this forms the basis of a useful test for putative dietary antioxidants. Recently, we have looked at the capacity of lymphocytes to recover from damage in vitro. Removal of H2O2 -induced strand breaks seems to be dependent on the level of antioxidants in plasma, and P-carotene apparently enhances cellular repair. However, the effect is probably a combination of antioxidant protection against continuing damage from atmospheric oxygen, and cellular DNA repair: results of such ex vivo experiments need to be interpreted with care. Session I: Oxidative DNA Damage and its Genetic Consequences
OXIDATIVE DNA BASE MODIFICATIONS AS FACTORS IN CARCINOGENESIS AND AIDS Ryszard Olinski, Department of Clinical Biochemistry, University School of Medical Sciences, Bydgoszcz, Poland
Reactive oxygen species (ROS) can cause extensive DNA modification including modified bases. Some of these DNA base damages have been found to possess premutagenic properties. Therefore, if not repaired, they can contribute to carcinogenesis. We have found elevated amounts of modified bases in cancerous and precancerous tissues when compared with normal tissues. Our data may indicate an important role of oxidative base damage in cancer initiation. Alternatively the increased level of the modified base products may contribute to genetic instability and metastatic potential of tumour cells. It is believed that patients infected with human immunodeficency virus (HIV) are under chronic oxidative stress. Moreover, recent evidence demonstrated that oxidative stress mediated by generation of ROS may be directly involved in apoptotic death of lymphocytes in HIV infected patients. Unrepaired DNA damage is one of the reasons of apoptosis. Therefore, it was interesting to analyse the modified base products in DNA isolated from lymphocytes of HIV infected individuals. We have shown statistically significant increase of oxidatively modified DNA bases in AIDS patients when compared with control group. In the case of one base product namely 8-oxo-G, which has a high mutational potential, statistically significant changes were also found between control group and asymptotic, HIV positive patients. We postulate that the observed increase in modified base level may be one of the causes responsible for apoptotic death of lymphocytes and cancers related to HIV infection. Session I: PL9800366 Oxidative DNA Damage and its Genetic Consequences
FORMATION OF IMIDAZOLE RING-OPENED PURINES IN DNA AND THEIR BIOLOGICAL CONSEQUENCES Barbara Tudek Institute of Biochemistry and Biophysics, Polish Academy of Scienes, Warsaw, Poland
Formation of imidazole ring-opened purines in DNA occurs spontaneously in the course of cellular oxidative processes as well as in the response to DNA alkylation with environmental pollutants or anticancer drugs. Accumulating data suggest that their presence in DNA may be both lethal and mutagenic. Fapy-7MeGua when present in DNA blocks replication by prokaryotic polymerases in vitro and in E.coli cells and causes 2-3-fold increase in G-»T and G—>C transversions. Treatment of single-stranded Ml3 phage DNA with dimethylsulfate and NaOH, in addition to Fapy-7MeGua formation, creates another mutagenic derivative, which gives rise to 60-fold increase in A—»G transitions in the lacZ gene of the phage. We have observed that selective depurination of 7MeAde prior to imidazole ring opening in alkali (as measured by HPLC chromatography of [3H]MNU alkylated DNA) coincided with 10-fold decrease of the frequency of A—>G transitions. This suggested that imidazole ring opening of 7MeAde might occur in alkali to give rise to a potent miscoding lesion Fapy-7MeAde. Hydroxyl radicals induce oxidation and imidazole ring opening of unsubstituted purines. We have studied in vitro DNA synthesis on oxidized templates containing Fapy- Gua, 8OH-Gua and Fapy-Ade as quantitatively predominant lesions, followed by thymine glycol and other oxidized pyrimidines. Using 3 different DNA polymerases: T7 DNA polymerase, Klenow fragment of DNA polymerase I from E.coli and DNA polymerase P from calf thymus we have observed that DNA synthesis was blocked by lesions in a sequence dependent manner. For prokaryotic polymerases (T7, Klenow) oxidized pyrimidines and for polymerase p oxidized purines were the major stop sites, respectively. Polymerase P showed a tendency to stop replication opposite oxidized purine adjacent to pyrimidine, while polymerase T7 and Klenow fragment more easily bypassed the same sequence. For all 3 polymerases oxidized purines more efficiently stopped replication when present in the middle of purine tract containing Ade and Gua. These results suggest that lethal properties of unsubstituted formamidopyrimidines (Fapy-Gua, Fapy-Ade) might depend on the surrounding nucleotide sequence and on the properties of DNA polymerase. The potential mutagenic activity will be discussed. Session I: Oxidative DNA Damage and its Genetic Consequences
ESTIMATION OF OXIDATIVE DAMAGE IN MAN FROM URINARY EXCRETION OF REPAIR PRODUCTS. Steffen Loft and Henrik E. Poulsen. Department of Pharmacology, Panum Institute, University of Copenhagen and Department of Clinical Pharmacology, University Hospital Copenhagen, Denmark
DNA is constantly damaged and repaired in living cells. The repair products of the oxidative DNA lesions, i.e. oxidized nucleosides and bases, are poor substrates for the enzymes involved in nucleotide synthesis, fairly water soluble, and generally excreted into the urine without further metabolism. Among the possible products 8-oxodG, 8- oxoGua, Tg, dTg and 5-OHmU, have so far been identified in urine. The excretion rates correspond to up to 104 oxidative DNA modifications in each cell every day. Given this high rate and the slow accumulation of lesions with age, repair efficiency must excess 99% in accordance with the short half-life of 8-oxodG in irradiated animals. It should be emphasized that the excretion of the repair products in urine represents the average rate of damage in the total body whereas the level of oxidized bases in nuclear DNA a concentration measurement in that specific tissue /cells in the moment of sampling. Interestingly, in a rat experiment we found that oxidative stress caused by 2-nitropropane increased urinary excretion of 8-oxodG to an extent that completely corresponded to the excess of nuclear 8-oxodG levels induced mainly in the liver and kidney. The rate of oxidative DNA modifications has been studied in humans by means of the repair products as urinary biomarkers, particularly with respect to 8-oxodG. The data obtained so far indicate that the important determinants of the oxidative damage rate include tobacco smoking, oxygen consumption and some inflammatory diseases whereas diet composition, energy restriction and antioxidant supplements have minimal influence possibly with the exception of yet unidentified phytochemicals, e.g. from cruciferous vegetables. The data are consistent with the experimentally based notion that oxidative DNA damage is an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still warranted. In future the use of the biomarkers may provide this evidence and allow further investigation of the qualitative and quantitative importance of oxidative DNA modification and carcinogenesis in man as well as elucidate possible preventive measures. Session I: Oxidative DNA Damage and its Genetic Consequences
FORMATION OF DNA ETHENO ADDUCTS IN RHODENTS AND HUMANS AND THEIR ROLE IN CARCINOGENESIS Alain Barbin. International Agency for Research on Cancer, 150, Cours Albert Thomas, 69372 Lyon Cedex 08, France.
Etheno adducts are exocyclic adducts formed with the nucleic acid bases adenine, cytosine and guanine by some environmental carcinogens such as vinyl chloride or urethane. In the last few years, they have received a renewed interest, due to the development of sensitive techniques of analysis that made it possible to measure their formation in vivo. This minireview summarizes the information gained recently from the work of several laboratories including ours. Increased levels of DNA etheno adducts have been measured in target tissues from rhodents exposed to vinyl chloride or urethane. Hepatic tumours induced by vinyl chloride in humans and in rats exhibit base pair substitution mutations in the ras and p53 genes, these mutations are consistent with the promutagenic properties of ethenobases. However, background levels of etheno adducts have been detected in DNA from non-exposed humans or animals, pointing to an alternative, endogenous pathway of formation. This background may be affected by dietary factors. It could arise from the reaction of /ra/w-4-hydroxy-2- nonenal (or its epoxide 2,3-epoxy-4-hydroxynonanal), a lipid peroxidation product, with nucleic acid bases. Elevated levels of etheno adducts are found in hepatic DNA from humans and rodents with genetic predisposition to oxidative stress and lipid peroxidation in the liver, and with an associated increased risk of liver cancer. Etheno-DNA adducts along with other oxidative DNA base damages may thus be involved in the induction of hepatocarcinogenesis in LEC rats and in patients with metal-ions storage diseases. These data indicate that ethenobases in DNA could serve as new biomarkers of oxidative stress/lipid peroxidation. Session I: Oxidative DNA Damage and its Genetic Consequences
HYDROGEN PEROXIDE EFFECTS IN Escherichia coli CELLS Nasser R. Asad1, Lidia M.B.O. Asad1, Andre B Silva2,1. Felzenszwalb' and Alvaro C. Leitao2 'Universidade do Estado do Rio de Janeiro, Instituto de Biologia, Rio de Janeiro, 20550-030, Brazil 2Universidade Federal do Rio de Janeiro, Instituto de Biofisica, Rio de Janeiro, 2194-590, Brazil
Several studies indicate that hydrogen peroxide (H202) causes different effects in E. coli cells. These effects are dependent on iron conditions and H2O2 concentrations. In this work we analysed the lesions produced in low iron conditions, the cross adaptive response and the synergistic lethal effect produced by o-phenantroline. In normal iron conditions the lesions produced by H202 are mainly repaired by the exonuclease III protein. In low iron conditions we verified that the Fpg and UvrA proteins and the SOS and OxyR systems participate in the repair of these lesions produced by H202. The o-phenantroline enhances the lethal effect of H202, if both are added simultaneously to the culture medium. We suggest that the kinetics of reaction of o-phenantroline with intracellular Fe+2 can enhance hydroxyl radical production by H202. E. coli cells treated with low concentrations of H2O2 exibit protection against lethal effects of different agents (cross adaptive response). Our results indicate that pretreatment with high H202 concentrations protects the cells against UV killing, an effect independent of the SOS system, but dependent on RecA and UvrA proteins. H202 also induces protection against the lethal and mutagenic effects of N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). This effect may be due to a modification of the MNNG alkylation pattern in the oxidized DNA. H202 also protects the cells against cumene hydroperoxide killing. This response possibly involves the alteration of the electrophoretic profile of the smaller subunit (22-kDa) of alkyl hydroperoxide reductase (Ahp). Session I: Oxidative DNA Damage and its Genetic Consequences
OXIDATIVE DNA DAMAGE AS A CAUSE OF MUTATION IN NON-DIVIDING CELLS. Brvn Bridges and Andrew Timms, MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, BN1 9RR, Gt. Britain
Bacterial cells that are starved and are not dividing, or are dividing extremely slowly, can nevertheless mutate. Given that most cells in nature are in this state it is surprising that the mechanisms involved have only recently attracted attention. It has become clear that the pathways involved and the spectra of mutations that arise in stationary phase bacteria are both clearly different from those observed in populations of growing bacteria. We have shown that glycosylases able to remove 8- oxyguaniane or adenine that is mispaired with 8-oxyguanine are intimately involved. Deficiencies in those enzymes result in considerable enhancement of the mutation rate and their overproduction results in a lowering of the rate. In stationery phase cells, 8-oxyguanine: adenine mispairs and other base mispairs give rise to small deletions in addition to the expected transitions and transversions found with growing bacteria. Evidence will be presented that oxidative species such as singlet oxygen occur in stationary phase cells and that miscoding base damage rise to a transient mutant phenotype that can trigger replication and lead to a fixed mutation in DNA. PL9800367 Session I: Oxidative DNA Damage and its Genetic Consequences
REPAIR OF IONIZING RADIATION DAMAGE BY DNA AND DEOXYNUCLEOSIDES R. C. von Borsteh John A. Higgins, and Ursula G.G. Hennig Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6H 2E9, Canada
The Centennial of the discovery of Polonium and Radium is also the Centennial for a more obscure event, the discovery by Ames and Huntley that Miescher's "nuclein", now called DNA, could enhance hematopoiesis. The first suggestion that a "humoral substance" from cell extracts could reverse lethal exposures of mice to ionizing radiation was accomplished by Kelly and Jones in 1953, followed by Cole and Ellis showing that deoxyribonuclease or trypsin inactivated this humoral substance. In 1958, Kanazir and his colleagues demonstrated clearly that DNA injected into rats could reverse lethal radiation exposures, but deoxyribonucleosides did not enhance survival. In 1965, Wilczok and Mendecki showed, as DNA is degraded into smaller oligodeoxy- nucleotides, the degraded products become less able to repair lethal damage. Contrary to findings in intact rodents, Petrovic and his collaborators demonstrated that four deoxyribonucleosides strongly enhance survival of irradiated cells. In 1973, Beltz and his colleagues demonstrated that deoxyribonucleosides are broken down rapidly in blood in vitro, and disappearance of deoxyribonucleosides is even more rapid in vivo. Thus the paradox of DNA repair and nucleoside nonrepair in whole animals is resolved. Our laboratory has shown that deoxyribonucleosides are nearly as effective as DNA in reversing chromosomal damage in intact animals, while DNA provides much better survival. Possible interactions of deoxyribonucleosides with other components of DNA repair will be discussed.
10 Session I: Oxidative DNA Damage and its Genetic Consequences
Prof. David SHUGAR
11 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
STRUCTURE-FUNCTION RELATIONSHIPS IN REPLICATION AND REPAIR OF CARCINOGEN-DAMAGED DNA B. Singer, B. Hang, J. Sagi and A. Chenna Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720
Exocyclic DNA adducts can be produced by either exogenous carcinogens or endogenous chemicals arising from normal metabolism and oxidative stress. Such adducts, without repair, can be mutagenic. Our laboratory has previously reported that etheno derivatives formed from chloroacetaldehyde are repaired by base excision. However, the structurally closely related 1 ,N6-etheno-A (e-A) and 3,N4-etheno-C (e-C) are excised by separate glycosylases. In this work, we investigated the human and E. coli enzymatic repair activities toward the exocyclic benzoetheno derivatives produced by benzene metabolite, p- benzoquinone (pBQ), using defined 25-mer oligomers. A 38,1 kDa human protein with a 5' AP endonuclease activity was isolated and proved to be the major human AP endonuclease, and found to directly incise oligomers containing 3, N4-benzentheno-2'-dC (pBQ-dC) 5' to the adduct without prior generation of an AP site, resulting in the adduct left as a ,,dangling base" on the 5' terminus. This mechanism is also utilized by two E. coli 5' AP endonucleases, exo III and endo IV, for the processing of the pBQ-dC adducts in oligomers. In contrast, we did not find any measurable nicking activity in human cells toward another pBQ derivative, l,N2-benzetheno-2'-dG (pBQ-dG). However, pBQ-dG was incised by exo III and endo IV with the same unusual mode of action. Neither pBQ-dC nor pBQ-dG are substrates for the E. coli AP lyase (3' AP endonuclease) - containing Fapy-DNA glycosylase. The presence of these different mechanisms of enzyme specificity for the repair of exocyclic DNA adducts poses important questions on structure-function relationships in molecular enzymology, in as much as molecular modeling and thermodynamics indicate that gross distortion occurs. Nevertheless, a low level of in vitro misincorporation is demonstrated for some of these oligomers.
12 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
AUTONOMOUS POLYMERASE FIDELITY John W. Drake National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
DNA-based microbes for which reliable information is available display a common rate of spontaneous mutation of about 0.0033 per genome per genome replication. (Recently, an extremophile appears to have joined this list.) Mutation rates per average base pair are reciprocally related to genome size over a range of about 6500-fold. The major determinants of microbial mutation rates are the accuracy of polymerization, the ability of the polymerase to extend from mispairs, the efficiency of proofreading, and the efficiency of several DNA mismatch repair functions; DNA excision repair and pool-cleansing functions may also contribute. The first three steps are often conducted by a DNA polymerase and its associated 3'-exonuclease, which may reside in the same replicase polypeptide. In addition, up to 20 or 30 other proteins may be closely linked to DNA replication, and several of these interact directly with the replicase. Structural studies suggest that the polymerase active center is somewhat interior to the molecule. This raises the question of whether replicase accuracy resides mainly within the replicase itself, or depends (as do processivity and velocity) upon additional proteins. This question has now been addressed experimentally.
13 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
STRUCTURE - FUNCTION STUDIES OF DNA POLYMERASES Katarzyna Bebenek'. Deborah A. Lewis', William A. Beard2, Thomas A. Darden3, Leping Li3, Rajendra Prasad2, Bruce A. Luxon4, David G. Gorenstein4, Samuel H.Wilson2 and Thomas A. Kunkel1'2 'Laboratory of Molecular Genetics, laboratory of Structural Biology, 3Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, 4Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555.
The objective of this study is to understand the interactions between DNA polymerases and their substrates that determine the ability of these enzymes to discriminate against errors. Three DNA polymerases for which structural information is available, Klenow fragment of E.coli Pol I, DNA polymerase p and HIV-1 reverse transcriptase (RT), are used as model systems. Here results for HIV-1 RT will be discussed. Molecular dynamics modeling based on the high resolution structure of apoHIV-1 RT and the lower resolution structure of a HIV-1 RT-DNA co-crystal suggests that five amino acids that interact with the duplex template primer comprise a track-like structural element, the minor groove binding track (MGBT). Changing any of these five residues to alanine yields RT with lower DNA binding affinity, lower processivity and altered fidelity for errors initiated by strand slippage in homopolymeric runs. These results indicate that both processivity and frameshift fidelity are controlled by the interactions of the enzyme with the template-primer upstream from the active site. We are also characterizing the properties of RT mutants containing single amino acid substitutions to alanine around the active site. Based on structural and biochemical information on HIV-1 RT and other DNA polymerases, we believe that the targeted residues contribute to interactions with the primer terminus, the incoming dNTP, and the active site template nucleotide and/or single strand template nucleotides. Characterization of the fidelity of several of these mutant RTs will be discussed.
14 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
UNEQUAL FIDELITY OF LEADING AND LAGGING STRAND REPLICATION ON E. COLI CHROMOSOME Iwona J. F ij alkowska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
The mechanisms by which organisms produce mutations, including errors of DNA replication, are of considerable interest. During chromosomal DNA replication, the two DNA strands are replicated asymmetrically, one strand being synthesized continuously (leading strand), the other discontinuously in the form of short ,,Okazaki" fragments (lagging strand). We investigated whether replication errors occur at different rates during leading and lagging strand DNA replication on the Escherichia coli chromosome. Strains were constructed in which the lac operon, derived from DNA of bacterial strains carrying several defined lacZ alleles was inserted into the chromosome in two different orientations with respect to the chromosomal origin of DNA replication. Measurement of lacZ reversion frequencies in strains defective in DNA mismatch repair or in exonucleolytic proofreading, in which mutations represent replication errors, indicated that there are distinct differences in the DNA replication fidelity between the two DNA strands. In contrast to the previous conclusions from plasmid based systems, we can present arguments that on the E. coli chromosome lagging-strand replication may be more accurate than leading strand replication.
15 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
MUTAGENESIS AND DNA REPLICATION FIDELITY IN ESCHERICHIA COLL Roel M. Schaaper, Jin-Yao Mo, and Phuong T. Pham National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
Duplication of the E. coli chromosome proceeds with high accuracy, the error rate being as low as 10"'° errors per bp per round of replication. Chromosome duplication is performed by pol III holoenzyme (HE), a large dimeric complex (18 subunits) that simultaneously replicates the leading and lagging strands at the replication fork. From an analysis of mutation rates in mismatch-repair-proficient and -deficient strains, we estimate the intrinsic accuracy of pol III HE in vivo to be about 10"7 (with DNA mismatch repair contributing the additional three orders of magnitude). In order to understand how HE can achieve its high accuracy (107), we have initiated in vitro fidelity studies with the catalytic alpha subunit (dnaE gene product), as well as various HE subassemblies, including the complete HE. Studies with the alpha subunit revealed an unexpected specificity: essentially no base substitutions, but many -1 frameshifts. The absence of base sub- stitutions does not reflect high accuracy at the misinsertion step but the inability to extend from mismatched termini. The enzyme has a high propensity to misalign these termini if the sequence permits slippage of the incorrect base on the next template base, accounting for the high frequency of frameshifts. We are currently investigating whether the unusual specificity of alpha subunit is altered by the additional subunits present in the pol III core or HE.
16 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
STEPS IN TRANSLESION SYNTHESIS OF REPLICATION BLOCKING LESIONS. Rita L. Napolitano and Robert P. P. Fusch UPR 9003 du CNRS, Cancerogenese et Mutagenese Moleculaire et Structurale, ESBS, Blvd S. Brant, 67400 Strasbourg, France
Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In E. coli, efficient translesion synthesis of a replication blocking lesion requires the induction of SOS-controlled gene products including recA and umuDC. To study translesion synthesis in vivo, we have constructed single stranded vectors containing single N-2-acetylaminofluorene (AAF) adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non- SOS induced conditions. Induction of the SOS response led to a ten fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly, we find that error-free translesion synthesis depends upon functional umuDC* gene products while mutagenic translesion synthesis is umuDC* independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication blocking lesion.
17 PL9800368 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
SOLUTIONS TO REPLICATION ON DAMAGED DNA TEMPLATES Shoshana Squires and Robert T.Johnson Department of Zoology, Cambridge University, Cambridge CB2 3EJ, United Kingdom
In recent years great advances have been made in our understanding of mechanisms of DNA repair in mammalian cells. There has been much less progress, however, in the study of mechanisms that influence replication on damaged templates (the field of post-replication repair), despite the biological and clinical importance of this subject. We have reinvestigated the response of active replicons in human cells to ultraviolet (UVC) light and have identified for this fraction of the genome two complementary mechanisms used by S-phase cells to protect the genome from the destabilizing effects of UV irradiation. These mechanisms are: a prolonged replication stalling at UVC-induced cyclobutane pyrimidine dimers (CPDs), and an enhanced repair of these lesions in the replicons active at the time of irradiation. Using neutral pulse field and alkaline gel electrophoresis techniques we find that stalling is very rapid, UV dose-dependent and prolonged: the stalled replication machinery does not appear to bypass the lesions. Recovery from stalling depends on CPD removal and the rate of repair in replicons active at the time of irradiation is two to four times that observed in the genome overall i.e. it is quantitatively similar to the preferential rate of repair in transcribed genes. In circumstances when many dimers remain several hours after UV, either after high UV fluences or in repair defective cells, the long-lived stalled sites are converted into cellular double strand DNA breaks, the frequency of which shows a close correlation with cell killing by UV.
18 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
YEAST GENES AND PROTEINS CONCERNED WITH TRANSLESION REPLICATION AND DNA-DAMAGE INDUCED MUTAGENESIS John Nelson1, Peter Gibbs1, David Hinkle2 and Chris Lawrence1, 'Department of Biochemistry & Biophysics, University of Rochester Medical Center, Rochester NY, 14642, USA, department of Biology, University of Rochester, Rochester NY 14627, USA.
Most spontaneous, as well as induced, mutations are likely to arise during replication past sites of DNA damage, and information about this process in eukaryotes may therefore provide novel strategies for the prevention of cancer. In budding yeast, translesion replication is performed by a non-essential enzyme called DNA polymerase £, which appears to carry out no other replication, recombination, or repair function. This enzyme is composed of a catalytic subunit encoded by the REV3 gene and at least one other subunit, the Rev7 protein. The Revl protein is also required for translesion replication in vivo. In keeping with its proposed function, we find that pol t, replicates past a T-T dimer or an abasic site in vitro more efficiently than yeast pol a. The enhanced efficiency depends, in part, on the relative insensitivity of pol C, to the structure of the DNA duplex at the end of the primer. This was examined by determining primer extension efficiency for two kinds of primer/template. In one, the primer abutted the T-T dimer or overlapped the dimer by 1, 2, or 3 nucleotides. In the other, dimer-free templates were used, but the primer ended in one or other of all possible mismatched and matched single base-pairs. The Revl protein possesses at least two functions: a general function facilitating translesion replication, possibly dependent on the formation of a transient complex with pol £, and a deoxycytidyl transferase activity that selectively inserts dCMP opposite abasic sites. Supported by NIH grant GM21858.
19 PL9800369 Kepair in Prokaryotes and Mechanisms of Mutagenesis
THE MECHANISM OF SOS MUTAGENESIS INVOLVES DYNAMIC INTERACTIONS BETWEEN RECOMBINATION AND MUTAGENESIS PROTEINS Adriana Bailone, Suzanne Sommer, Raymond Devoret, Francois Boudsocq, Severine Delacour, Institut Curie, Batiment 110, F-91405 ORSAY, France.
It is now clear from some of our recent work in E. coli that three main repair pathways are sequentially expressed after a UV-light flash damaging DNA: (1) Excision repair removes 80-85% of all lesions within 20 min of incubation in rich medium; (2) then, homologous recombination occurs in the next 40 min, leaving aside 60% of the lesions remaining after excision repair. DNA strand exchange fills the 1.8 kb DNA single-stranded gaps left downstream a lesion; (3) the gaps still left are now resolved by error-prone replication (SOS mutagenesis) during the next hour with a 50% efficiency. The successive pathways are triggered by sequential induction of several proteins, to name a few: UvrA and UvrB for excision repair, RecA protein for recombinational repair, and UmuD and UmuC for SOS mutagenesis. RecA is the very protein that starts this sequential induction by oligomerizing on single- stranded gapped DNA. RecA oligomerization entails rapid cleavage of LexA protein and a tardy UmuD cleavage giving rise to UmuD', the matured form of UmuD. The time course of LexA repressor inactivation, on the one hand, and LexA specific binding to various promoters of SOS genes, on the other hand, determine the kinetics of expression of SOS genes. The stronger the binding of LexA to an SOS gene, the tardiness of its expression. For example, the cell division gene sfiA is expressed very late. The UmuD'2C complex whose formation is indispensable for SOS mutagenesis appears late, slowly and parsimoniously.
Interestingly, we found that the UmuD'2C complex inhibits homologous recombination independently of any damage sustained by DNA. Recombination inhibition in Hfr x F' crosses by an artificially produced high cell concentration of the UmuD'2C complex may result from direct inhibition of RecA polymers by the complex. Thus, the UmuD'2C complex is a molecular switch that turns off recombinational repair while it turns on SOS mutagenic repair. RecA polymer behaves as an accordion on single-stranded DNA generated by a lesion. First, RecA monomers are produced at a high rate and RecA polymers expand, enhancing recombinational repair. The UmuD'2C complex may bind at the end of RecA polymers preventing recombinational strand exchange. RecA polymers act as a chaperone positioning the UmuD'2C complex at a lesion that will be sensed by DNA polymerase. Then, DNA polymerase resumes replication in an error-prone manner across a lesion. RecA is pushed away from the single- stranded DNA. The DNA double helix is restored while there may be changes in amino acid coding.
In conclusion, the UmuD'2C complex is a timely molecular regulator that switches repair from recombination to error-prone replication.
20 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
STRUCTURAL INSIGHTS INTO THE REGULATION OF SOS MUTAGENESIS Martin Gonzalez, Ekaterina G. Frank, John P. McDonald, Mary McLenigan, Arthur S. Levine, and Roger Woodgate Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2725 USA
The Escherichia coli Umu proteins are best characterized by their role in damage inducible mutagenesis. Recently, we have discovered that the intracellular levels of the UmuD and UmuC proteins are kept to a minimum by the Lon serine protease. Studies with the Salmonella typhimurium UmuD protein (which is 73% homologous with its E. coli counterpart), revealed that it too is degraded by Lon and suggests that both UmuD proteins share conserved structural motifs. In contrast, E. coli UmuD' is removed from the cell by the ClpXP serine protease, but only when it is in a heterodimer complex with UmuD. We have generated deletion mutants of UmuD' and have co-expressed the mutant protein with UmuD. By assaying the sensitivity of the mutant UmuD'- UmuD complex to ClpXP, we have been able to map residues of UmuD' that appear essential for efficient UmuD'-UmuD heterodimer formation. Previous experiments have suggested that the in vivo posttranslational processing of UmuD to UmuD' is inefficient. We have, however, discovered that limited cleavage occurs in an undamaged cell, but that these small amounts of UmuD' are rapidly degraded by ClpXP, thus giving rise to the appearance of inefficient cleavage. The ClpXP protease therefore plays dual roles in regulating SOS mutagenesis: it keeps the basal levels of UmuD' to a minimum in undamaged cells but it also acts in damaged cells to reduce the elevated levels of mutagenically active UmuD' protein thereby returning the cell to a resting non-mutable state.
21 PL9800370 c . TT Session II: LUNA Repair in Prokaryotes and Mechanisms of Mutagenesis
A NEW GENE IN Escherichia coli K12, isfA, MAY BE INVOLVED IN DOWN REGULATION OF THE SOS SYSTEM. Anna Bebenek Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
The isfA mutation was discovered in one of the two ApolA strains isolated by Joyce and Grindley, Ci21%ApolA, which was characterized as the ApolA strain able to grow on rich medium. We have found that the ability to grow on rich medium coincided with antimutator activity and was related to unknown mutation - denoted later as isfA (Inhibition of SOS Function). The mutation was mapped at 85 minute on the E. coli chromosome and was shown to be responsible for inhibition of several phenomena related to the SOS response, also in polA+ strains. These phenomena include: UV and MMS-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and DNA repair. Mutator activity in the strains constitutivelly expressing RecA coprotease, recA730 and recA730lexA(Def), was also significantly decreased by the isfA mutation. The overall results suggesting that the product of the mutated isfA gene may be involved in the inhibition of RecA coprotease activity were supported by the immunobloting analysis showing that in recA730lexA(Def) isfA cells, the processing of UmuD protein to UmuD' was inhibited. The isfA mutation causes selective inhibition of the SOS-dependent mutagenesis and other SOS- functions, but not recombination. The hypothesis is proposed that the isfA gene product may negatively control the SOS system, and provides the mechanism for it's down regulation to allow the cell to return to its normal state.
22 o . TT PL9800371 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
STUDIES ON UV-INDUCED MUTAGENESIS NOT RELATED TO DNA REPLICATION. Irena Pietrzvkowska and Anna Czajkowska, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
We have previously shown that UV-induced mutations in E. coli uvrA and uvrArecF are formed very early after irradiation. This suggested that mutations may arise in a way independent of damaged DNA replication. This was further supported by studies on XsusOg phage, an amber mutant, unable to replicate its DNA in su host. We have found that UV-induced reversion of 'ksusOs to sus+ may occur in the su host nonpermissive for phage DNA replication. This mutagenic pathway is independent of UvrABC-excinuclease activity and requires a high level of UmuD' and basal, noniduced, level of UmuC. This mutagenic pathway is different from that occurring in su+ cells permissive for phage DNA replication. Deletion of utnuDC operon completely suppresses UV-induced mutagenesis of XsusO8 in su host cells but not in su+. The difference in the mutagenic pathways of XsusO8 under permissive and non permissive conditions for phage DNA replication will be discussed.
23 PL9800372 _ . TT Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
EFFECT OF TRANSPOSON Tn/0 ON THE SURVIVAL AND MUTATION FREQUENCY OF HALOGEN LIGHT-IRRADIATED E. co//K-12 STRAIN AB1157 Celina Janion and Anna Wqjcik Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland
Transposons are widely used as tools for constructions of bacterial strains and as carriers for genetic material. We have found that Tn/0 transposon, when inserted into chromosome of E. coli strain AB1157, renders the cells more sensitive to and less mutable by halogen light irradiation. These effects are not dependent on the chromosomal locus from which Tn/0 derives or at which is inserted. Halogen light contains a broad spectrum of UV radiation (UVA, UVB and UVC), and both its potency and specificity of mutation results from formation of pyrimidine dimers and 6-4 photoproducts in DNA. The induced mutations are UmuD, UmuC-protein dependent. We believe that the effects of Tn/0 on survival and mutation frequency in halogen light- irradiated bacteria (tested as argE3 to Arg+ reversion) is a result of UmuD and UmuC depletion, since: (i) the decline in survival is about the same in Tn/0-bearing bacteria as in bacteria with deleted umuDC; (ii) transformation of Tn/0 bearing bacteria with plasmid overproducing UmuDC or UmuD'C leads to increase in survival and mutation frequency; and (iii) insertion of Tn/0 into chromosomal DNA has no effect on the frequency of mutations induced by EMS, a mutagen which activity is umuDC independent. However, transformation of bacteria deleted in umuDC with plasmids carrying umuDC or umuD 'C leads to recovery of mutability, but not of survival of halogen light irradiated cells, indicating that the mechanisms leading to wmw£>C-dependent mutagenesis and to w/wwZ)C-dependent protection of survival must be different.
24 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
MECHANISMS OF MUTAGENESIS BY CYTOSINE ANALOGS Hikoya Hayatsu1 and Kazuo Negishi2 'Faculty of Pharmaceutical Sciences, and 2Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan
Mutations occur by base structure changes in DNA. Relations between a certain type of such a change and its resulting mutation specificity could offer insights into mutagenesis mechanisms in general. Nucleobase analog-induced mutagenesis provides an opportunity for such an insight. We have discovered that N(4)aminocytidine, a nucleoside analog bearing an extra amino group on the N(4)amino moiety of cytidine, is a potent mutagen towards phages, bacteria, mammalian cells and fruit flies. Our studies on the mechanisms of this mutagenesis, using mainly the E. coli systems, have revealed that (1) N(4)aminooytosine is incorporated into DNA during the process of replication (2) during that process this analog may be incorporated as a substitute of either cytosine or thymine, thereby causing a C to T transition, and (3) this analog as a constituent of the template DNA may direct incorporation of either guanine or adenine, thereby causing a G to A transition. This ambivalent nature of N(4)aminocytosine may be derived from a tautomerism between its amino and imino forms, with the amino form basepairing with guanine and the imino form with adenine. A recent finding in our laboratory is that Ni2+ ion can enhance significantly the mutagenic potency of N(4)aminocytidine. This phenomenon is probably related to the fact that Ni2+ forms a tight complex with N(4)aminocytidine. Overall, the structural alteration in cytosine, namely, the presence of an additional amino group at the N(4)amino moiety, results in a high mutability to cause specifically transitions.
25 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
MUTAGENIC PATHWAYS OF THE BASE ANALOG, 6-W-HYDR0XYLAMIN0PURINE, IN BACTERIA AND YEAST Youri I.Pavlov Universitetskaya emb. 7/9, Department of Genetics, Sankt-Petersburg State University, Sankt-Petersburg, 199034, Russia
Base analog 6-JV-hydroxylaminopurine (HAP) is a potent mutagen in variety of prokaryotic and eukaryotic organisms. HAP mutagenesis is not affected by defects in generalized repair mechanisms - mismatch, excision, mutagenic and recombination repair, but is elevated several- fold in the DNA polymerase mutants with defective exonucleolytic proofreading in bacteria and yeast, suggesting that HAP-induced errors could be partially eliminated by editing function of DNA polymerases. To investigate this effect in more detail we designed in yeast a special system of strains which are viable despite being completely proofreading-defective, possessing defects of exonuclease sites of both replicative DNA polymerases, delta and epsilon. Antimutator mutations impairing polymerase III function in E. coli strongly suppressed HAP mutagenesis. In yeast, mutations affecting any of three replicative DNA polymerases, PCNA or RF-C were HAP-antimutators. We found that mutants in the yeast AAH1 (adenine aminohydrolase) gene increase HAP mutagenesis, suggesting that this enzyme plays a role in HAP detoxification. Another gene, HAM1 (HAP-mutability) gene, in which mutations confer HAP hypermutability, has homologs in the wide range of organisms, from E.coli to C.elegans and man. The homologous E. coli gene is located in the 65-68 min region of the chromosome, while we mapped a newly obtained point mutation conferring HAP hypermutability to 18.7 min. Thus, a suprisingly large number of genes control HAP mutagenesis, raising the possibility that this base analog might pose an everyday threat to the cell.
26 Session II: DNA Repair in Prokaryotes and Mechanisms of Mutagenesis
TEMPLATE - DIRECTED BASE PAIRING OF 2-CHLORO-2'-DEOXYADENOSINE CATALYZED BY AMV REVERSE TRANSCRIPTASE Jaroskw T. Kusmierek and Agnieszka M. Bukowska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
2-Chloro-2'-deoxyadenosine (2C1A) is used for treatment of several lymphoid malignances. It is known that 2C1A is incorporated into DNA in cells. The aim of our study was to determine the coding properties of 2C1A located at preselected sites in oligonucleotide templates. 2C1A phosphoramidite was prepared and was used for synthesis of 25-mer templates with 2C1A located at site 21 from the 3'-end. Kinetic parameters (Km and Vmax) for site specific incorporation of dNTPs by AMV reverse transcriptase were determined. Frequency of incorporation (Vmax/Kn,) of dTTP opposite 2C1A is 25 - 50 fold lower than opposite A in each of four sequences studied. Substitution of chlorine in adenine strongly influences the Km values (0.08 - 0.18 ^iM for A and 1.8 - 4.9 \iM for 2CIA), whereas Vmax values are similar, especially when these values for A and 2C1A are compared in the same sequence. The extension of 2C1A • T pair occurs with about 3 times lower frequency than extension of A • T pair. The frequency of insertion of dCTP, dGTP and dATP opposite 2C1A was at least several times lower than opposite A. The obtained results suggest that the presence of 2C1A in DNA template considerably slows DNA replication but does not lead to base substitution mutations.
27 PL9800373 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
GENETIC BASIS OF RADIATION SENSITIVITY IN MAMMALIAN CELLS Mafgorzata Z. Zdzienicka Department of Radiation Genetics and Chemical Mutagenesis-MGC, Leiden University, Wassenarseweg 72, 2333 AL Leiden, The Netherlands.
To dissect and elucidate the mammalian cellular response to ionizing radiation, X-ray-sensitive mutants have been investigated. In humans two hereditary disorders are shown to have clearly an increased sensitivity to ionizing radiation (IR): ataxia-telangiectasis (AT) and Nijmegen breakage syndrome (NBS). To gain more insight into the defect in NBS cells, two immortal fibroblasts cell lines were obtained and characterized. The transfer of a single human chromosome 8 into these cell lines complemented the defect: X-ray sensitivity and radioresistant DNA synthesis (RDS) confirming that the NBS gene is located on this chromosome. In cultured rodent cells several X-ray sensitive mutants have been isolated and at least eleven complementation groups identified [reviewed in Zdzienicka, Cancer Surveys, 28 (1996) 281-293]. Amongst these groups, one group appears defective in the gene homologous to the ATM gene. The hamster mutants, like AT cells, show RDS in response to IR. The complexity of the regulation of DNA replication in response to IR is indicated by the identification of additional genes responsible for RDS: one on human chromosome 4q and another defective in the hamster cell mutant (V-C8). In addition, several groups of IR sensitive mutants, which are impaired in repair of DNA double-strand break (DSB), have been isolated. Using these mutants components of the DNA- PK, complex have been identified. Molecular analysis of hamster Ku86 cell mutants and characterization of new mutants defective in DNA-PKCS will be presented.
28 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
STUDIES OF EUKARYOTIC DNA MISMATCH REPAIR Thomas A. Kunkel Laboratory of Molecular Genetics, NIEHS, Research Triangle Park, NC 27709 USA
Once cells have accurately replicated their genomes, rare errors left behind by the replication machinery are corrected by DNA mismatch repair. Defective mismatch repair is associated with several types of cancer. In order to identify human mismatch repair genes and understand how their gene products function, we are studying human tumor cell lines from a variety of tissue types. We have identified several lines that are defective in mismatch repair activity, are resistant to treatment with MNNG and have elevated mutation rates in microsatellite sequences and in the endogenous HPRT gene. These cells contain mutations in one or more mismatch repair genes, including hMSH2, hMSH3, hMSH6, hMLHl or hPMS2. The mismatch repair defects, MNNG resistance and microsatellite instability in these lines have been corrected by introducing the relevant wild-type human chromosome. These lines are being used to examine the biochemical and biological functions of mismatch repair in human cells and the connection between defects in these functions and tumorigenesis and resistance to chemotherapeutic agents. Emphasis will be on recent studies showing that i) different gene products repair base-base and insertion/deletion mismatches, ii) PCNA is required for mismatch repair at a step preceding the DNA synthesis associated with the mismatch repair process and iii) mismatch repair is defective in fibroblastoid cell lines derived from Werner Syndrome patients.
29 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
BIOCHEMISTRY OF G/T MISMATCH REPAIR IN HUMAN CELLS. Teresa Lettieri and Joseph Jiricny Institute for Medical Radiobiology, August Forel Strasse 7, CH-8028 Zurich, Switzerland
Many cancers display C to T mutations in CpG dinucleotides in the p53 tumor suppressor gene. These arise presumably by the deamination of 5- methylcytosine. We have shown that G/T mispairs associated with this hydrolytic event are corrected by a base-excision repair process, which is initiated by a thymine-specific mismatch DNA-glycosylase, TDG. The TDG gene has been mapped to chromosome 12q24, but so far no cancer- associated mutations have been detected in this locus. Are the C to T transitions in p53 really due to the malfunction of TDG or are other factors at play? We hope to answer this question by constructing mice that are lacking the TDG gene. We have been studying the G/T mismatch repair process in mammalian DNA. The removal of the mispaired thymine by TDG initiates a base excision repair process, such that the resulting apyrimidinic site is first cleaved on its 5'-side by AP-endonuclease. In a subsequent step, polymerase-p catalyses the removal of the baseless sugar-phosphate and then fills in the single nucleotide gap The remaining nick is sealed by a DNA ligase and we assume that it is DNA ligase III/XRCC1 complex which fulfills this role in the TDG-mediated repair. In order to ensure that G/T-specific repair process does indeed involve the above proteins, we are currently expressing them in heterologous systems with the goal of being able to reconstitute the entire pathway from purified recombinant proteins.
Wiebauer, K., Neddermann, P., Hughes, M.J. and Jiricny, J. (1993) The repair of 5-methylcytosine deamination damage, in: DNA Methylation: Biological Significance (eds. J.-P. Jost and H.-P. Saluz) Birkhauser Verlag, Basel, Boston, pp 510-522. Lettieri, T. and Jiricny, J. (1997) In: Base excision repair of DNA damage (ed. I.D. Hickson) Landes Bioscience, Austin, pp 45-66.
30 Session III: PL9800374 DNA Repair in Eukaryotes and its Relation to Human diseases
NUCLEOTIDE EXCISION REPAIR: IN VITRO ANALYSIS Bernard Salles1, Patrick Calsou, Philippe Frit1, Ruo-Ya Li2, Catherine Muller' and Christian Provot2 'IPBS UPR9062 CNRS, 205 route de Narbonne, 31077 Toulouse cedex- France; 2S.F.R.L, Berganton, 33 127 Saint Jean d'lllac, France.
The nucleotide excision repair (NER) which processes virtually all the DNA damage, can be summarized as two main steps: recognition/incision/excision of the lesion and DNA synthesis/ligation to restore strand continuity. R.D. Wood et coll. have reproduced the entire reaction in vitro with cell-free extract incubated in the presence of damaged plasmid DNA (1). During the repair synthesis step, incorporation of labeled deoxynucleotides permits the quantification of the repair activity. In order to analyze the early steps of the reaction, we have developped two strategies: (i) we have modifed the biochemical conditions of the in vitro repair assay in such a way that the incision reaction might be analyzed; (ii) we have designed a DNA adsorption procedure in sensitized microplate wells in order to detect the proteins involved in the repair complex by an ELISA-like reaction. We have reported previously an inhibition of NER activity, at the level of the recognition/incision/excision step when the damaged DNA substrate was linearized by comparison with a supercoiled plasmid DNA (2). This inhibition was shown to result from DNA-end binding of the Ku autoantigen, an heterodimer involved in double-strand break repair and V(D)J recombination. Using the microplate assay in which the DNA is adsorbed on a solid phase (3), we reported here that the inhibition of NER activity correlated with a reduced binding of DNA repair proteins on damaged DNA, probably affecting the initial recognition step. In vivo , the relevance of such a phenomenon could account, in a Ku-dependent manner, for cell sensitization to DNA-damaging agents (UV light) following treatment with clastogenic agents.
(1) Wood, R.D. (1996) Annu Rev Biochem 65, 135-167. (2) P. Calsou, P. Frit and B. Salles (1996), J. Biol. Chem, 271, 27601- 27607 (3) B. Salles, C. Provot, P. Calsou et al. (1995), Anal. Biochem., 232, 37- 42
31 PL9800375 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
PROCESSING OF STRUCTURALLY DIFFERENT DNA LESIONS BY NUCLEOTIDE EXCISION REPAIR L.H.F.Mullenders, A. van Hoffen, M. van Oosterwijk, Harry Vrieling, A.T. Natarajan, Albert A. van Zeeland, Leiden University, The Netherlands.
We have investigated the contribution of the global genome (GGR) and the transcription-coupled repair pathway (TCR) to the removal of structurally different DNA lesions. The repair kinetics of UV-induced CPD and 6-4PP as well as NA-AAF induced adducts dG-C8-AF and dG- C8-AAF were determined in (in)active genes in normal human, xeroderma pigmentosum group C and Cockayne's syndrome cells. Our results can be summarized as follows: * All bulky lesions investigated are targets for TCR. However, in the case of normal human cells the contribution of TCR to repair of 6-4PP, dG-C8-AAF and dG-C8-AF in active genes is minor. * Results of experiments with different UV-doses suggest that (i) TCR of UV-induced photolesions takes place in a processive way and that (ii) the significance of TCR for the removal of lesions depends on the dose. * CS cells are sensitive to both UV-light and NA-AAF. The UV sensitivity is explained by the lack of TCR of CPD in CS cells. However, the kinetics of repair of dG-C8-AF in normal and CS cells are the same; yet CS cells are unable to recover NA-AAF inhibited RNA synthesis. Therefore, it is questionable whether the absence of recovery of the transcription process in CS cells in the presence of DNA damage is due to a defect in TCR. An alternative model will be discussed.
32 Session III: PL9800376 DNA Repair in Eukaryotes and its Relation to Human Diseases
COMPARISSON OF REPAIR ACTIVITY IN DIFFERENT GENOMIC REGIONS George Russev, Peter Stoilov1, Lyubomira Chakalova1, and Stoian Chakarov2 'institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria 2Faculty of Biology, University of Sofia, Sofia, Bulgaria
We developed a quantitative assay to determine repair of structurally different DNA lesions at defined genomic sites. This assay depends on the fact that many different types of damage are repaired by the same nucleotide excision repair pathway which includes synthesis of short DNA fragments at the sites of damage. After exposure to damaging agents, cells are treated with 5-bromodeoxyuridine (BrdUrd) to label the regions undergoing repair with the presumption that regions that have been more efficiently repaired would incorporate more BrdUrd than regions that were less effectively repaired. In this way the abundance of the different sequences in the BrdUrd containing DNA would be a direct and quantitative measure for the repair rates of the corresponding regions. The abundance of the different DNA sequences is determined either immunochemically, or by quantitative PCR using the BrdUrd- containing DNA fraction as a template. In the latter case the amount of product was directly proportional to concentration of template. This approach was used to address the question whether DNA repair after UV irridation occurs in an uniform, random manner or with preferences for certain regions. We found out that there was a higher repair efficiency at the 5'-end of the (3-globin gene both in human and mouse cells.
33 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
DNA REPAIR DOMAINS Miroslav Pirsel Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia
It is generally known that DNA repair is heterogeneous over the genome. The heterogeneity results from functional and morphological properties of DNA, i.e. its metabolic activities and chromatin structure. DNA is bound to nuclear matrix at specific sites forming chromatin loops or domains which enable more efficient regulation of various prosesses including repair. In addition, there are several proteins known to participate both in repair and transcription and some of them function as transcription repair coupling factor (TRCF) - a protein complex recognizing RNA polymerase II blocked by damage. We do not know whether the efficiency of repair depends directly upon frequency with which RNA polymerase II encounters a lesion and is blocked or whether transcription of active gene opens the entire domain to preferential repair. Indeed, repair of active genes was found to be limited to repair domains. The size is approximately 50-80 kb, which is also the average size of chromatin loop. It is not clear what determines the boundaries of the repair domain. Domain can fit the size of transcription unit, but can be also several times larger than transcription unit. To explain more satisfactory the concerted action of preferential and genome overall repair we hypothesize that repair domain is a dynamic functional and structural region of DNA having potential to change its size and regulate the extent of repair.
34 c . TTT PL9800377 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
DNA ALKYLATION DAMAGE, REPAIR AND CLINICAL SIGNIFICANCE G. P. Margison Peterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, U.K.
Tumor resistance to the therapeutic effects of the nitrosoureas (e.g. BCNU, Muphoran) and the methylating agents (e.g. DTIC, Temozolomide) can be mediated by high levels of expression of O6- alkylguanine-DNA alkyltransferase (ATase) although other mechanisms can also be important. Sensivity to the toxic side effects of these agents is likely to be related to low levels of expression of ATase in normal tissues. Ionizing radiation induces the expression of ATase in normal tissues. The recent observation that this is dependent on functional p53 has indicated a possible strategy for increasing ATase activity in normal but not p53 mutant tumor cells, thereby increasing differential sensivity: initial results using a tumor xenograft system are encouraging. Alternatives for increasing the sensitivity of tumor cells to these agents include the use of ATase inactivating agents and, to increase the resistance of normal cells, ATase gene therapy. Combinations of these latter approaches using inactivating agent-resistant varsions of ATase also look promising.
Supported by the Cancer Research Campaign and the Medical Research Council.
35 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
INACTIVATION OF ALKYLTRANSFERASE TO ENHANCE CHEMOTHERAPY Karol Kleibl Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia,
Resistance of tumour cells to anticancer agents that produce adducts at the O6 position of guanine in DNA correlates with the expression of 06 alkylguanine alkyltransferase (ATase). O6-benzylguanine and related agents are able to inactivate human ATase and they are tested as chemotherapeutic adjuvants for enhancing the effectiveness of antitumour drugs. On the other hand the consequences of such a total ATase depletion for clinical purposes may be fatal for some sensitive physiological systems e.g. hematopoesis. To overcome this problem, the attempt to protect sensitive cells by targeted transfer of resistant ATase genes seems to be promising. ATases contain highly conserved amino acid sequences in the region of their active site. The enzymatic activity as well as the sensitivity to pseudosubstrates can be modified even by single amino acid change. Using site directed mutagenesis we constructed a version of human ATase gene coding for the protein resistant to O6-benzylguanine and its derivatives. The human ATase wild type gene as well as its mutated form were inserted in E.coli (ada,ogt) cells. The protecting effect of these genes on survival after treatment of cells with alkylating agents has been studied in in vivo and in vitro experiments. The use of inactivators sensitizes human ATase expressed in bacterial cells however the transformants harboring plasmid with simple point mutation in ATase gene were refractory to their effect.
36 Session HI: DNA Repair in Eukaryotes and its Relation to Human Diseases
REPAIR OF DNA ALKYLATION DAMAGE Timothy R. O'Connor Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010, USA
The Base Excision Repair pathway is one of two different repair pathways involving excision of damaged DNA, The first step in this repair pathway is the excision of a single damaged base from DNA by a DNA glycosylase. The coding sequences for human and murine DNA glycosylases (MPG proteins) removing 3-methyladenine and 7- methylguanine were cloned and the enzymes were shown to have a broad substrate range. The human gene is located on the short arm of chromosome 16 and we are currently studying the promoter region. The MPG protein is the major enzyme in mammalian cells responsible for the removal of 3-methyladenine and 7-methylguanine damage. We have investigated the excision of these bases both in vitro using end-labeled plasmids, and in vivo in normal human male fibroblasts using ligation- mediated PCR. In vitro data indicates that there is up to a 200-fold difference in the excision of 7-methylguanine bases by the human MPG protein depending uniquely upon the base sequence. The hetereogeneity in the in vivo repair of 7-methylguanines was observed over an 800 bp region including the promoter and the first exon of this gene. The correlation between in vitro and in vivo data shows that excision of 7- methylguanine is the rate limiting step for Base Excision Repair and that in the absence of protein-DNA interactions (e.g. transcription factors) the hetereogeneity in the repair of damaged DNA bases is dependent on the rate of excision by the DNA glycosylase.
37 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
MAMMALIAN CELLULAR RESPONSES TO DNA CROSS-LINKING AGENTS Florence Larminat1. Efterpi Papouli', Gilles Cambois', Corinne Lafon1, Annie Valette1, Malgorzata Z. Zdzienicka2 and Martine Defais1 1 Institut de Pharmacologie et de Biologie Structurale, UPR 9062 CNRS, Toulouse, France; 2 University of Leiden, Leiden, The Netherlands.
The major aim of our study is to characterize the cellular responses to cross-linking agents. DNA cross-links are produced by a number of agents including mitomycin C and cis-platinum. The genetic and biochemical complexity of the mammalian cellular defense mechanisms against cross-links is reflected by the existence of at least 8 complementation groups amongst rodent cell mutants defective in this response. We have examined the biological consequences of a mitomycin C (MMC) treatment on 2 different hamster cell mutants hypersensitive to cross-linking agents: VH4 and VC8 cells, and their parental cell line V79. First, we have quantified the gene-specific repair of the MMC-induced DNA interstrand cross-links. The efficiency of repair was similar in the 3 cell lines. Then, we have analyzed the cell-cycle distribution by flow cytometry after treatment with increasing doses of MMC. All the cells were transiently arrested in S phase and subsequently accumulated in G2. Parental cells recovered rapidly from this G2 arrest. However, treatment of mutant cells resulted in a dose-dependent increase of cells arrested in G2/M transition and prolongation of the G2 arrest. We have also examined the induction of apoptosis in the 3 cell lines after treatment with equitoxic concentrations of MMC. The apoptotic cells were detected 72 hours after treatment by TUNEL assay and flow cytometry. The amount of apoptotic cells was significantly higher in the mutants than in the parental cells. Our data suggest that the defective genes in VH4 and VC8 mutant cells may be involved in the regulation of the apoptotic cascade following cross-linking treatment.
38 PL9800378 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
THE ROLE OF P53 IN DNA REPAIR DEFICIENT SYNDROMES HYPERSENSITIVE TO UV LIGHT Leela Daya-Grosiean, Nicolas Dumaz, Sophie Queille, Christiane Drougard and Alain Sarasin. Molecular Genetics Laboratory, IFC1, CNRS, BP8, VILLEJUIF, FRANCE.
The ultraviolet (UV) component of sunlight is known to induce lesions on genomic DNA. Among the many responses of human cells following genotoxic stress is the nuclear accumulation of p53, a tumor suppressor protein, which plays an essential role in cell cycle control. There exist three distinct human hereditary diseases hypersensitive to UV light, xeroderma pigmentosum (XP), Trichothiodystrophy (TTD) and Cockayne's syndrome (CS) that carry mutations in genes involved in nucleotide excision repair (NER) and RNA transcrption. We have analysed cells from these syndromes and have shown that the lesions localized on the transcribed strand of active genes are responsible for the prolonged accumulation of the p53 protein in human cell nuclei following UV induced damage. This protein protects the cells from the deleterious effects of UV. In fact, p53 is a transcriptional factor whose accumulation induces the expression of genes such as WAFl and GADD45. The products of these genes inhibit the replication of damaged DNA and stimulate its repair. If the repair is not complete, replication of UV damaged DNA transforms the lesions into mutations. These mutations may be responsible of oncogene activation and tumor suppressor gene inactivation which promote the tumoral transformation of cells. Inactivation of the p53 tumor suppressor gene by solar UVB induced mutations is an early and essential step of skin carcinogenesis and may promote genetic instability in cutaneous cells which lead to skin tumor development. The carcinogenic effects of UV light are manifested by the highly cancer prone XP patients where we have shown that over 70% of the cutaneous carcinomas carry significant alterations of the p53 gene. All mutations are targeted at dipyrimidine sites, the hot spots for UV induced lesions and the majority are GC->AT transitions. Most significantly, over 70% of these are CC>TT tandem mutations considered to be the veritable signature of UV induced lesions. The majority of the mutations are due to the presence of unrepaired lesions remaining on the the non-transcribed strand of the p53 gene in the XP tumors. Indeed, this is the first demonstration of the existence of preferential repair in man.
39 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
INFLUENCE OF THE ONCOGENE SUPPRESSOR PROTEIN p53 ON THE SENSITIVITY OF TUMOR CELLS TO DNA TOPOISOMERASE INHIBITORS Annette K, Larsen' Celine Gobert and Andrzej Skladanowski Laboratory of the Biology and Pharmacology of DNA Topoisomerases, CNRS URA 147, Institut Gustave Roussy PR2, 94805 Villejuif Cedex, France Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Gdansk, Poland
DNA topoisomerase inhibitors are important antineoplastic agents used in the treatment of both leukemias and solid tumors such as breast, lung and colon cancers. Their clinical usefulness is limited by both natural and acquired tumor cell resistance. A major progress in recent years is the realisation that the expression of certain oncogenes and oncogene suppressors commonly associated with human tumors may have a profound effect on how tumor cells respond to antineoplastic agents. The oncogene suppressor protein p53 is a multifunctional stress protein activated by DNA lesions and prolonged inhibition of RNA synthesis. DNA lesions will induce a number of parallel, partially independent pathways affecting DNA repair, cell cycle progression and cell death, which all can be affected by p53. We have used isogenic cell lines that only differ in p53 function to determine the influence of p53 status on the sensitivity toward topoisomerase inhibitors. The results show that p53 induction has a clear, cell type-specific effect on the sensitivity of tumor cells toward this important class of antineoplastic agents.
40 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
RECOMBINATION, MUTATION, AND ONCOGENIC TRANSFORMATION CAUSED BY DYSFUNCTION OF THE DNA CLEAVAGE-RELIGATION REACTION OF TOPOISOMERASE II. Ronald Hancock Centre de Recherche en Cancerologie de l'Universite Laval, Quebec, Canada G1R2J6.
Topoisomerase II plays a central role in regulating the topological state of genomic DNA in vivo, and appears to continuously scan DNA for regions which require relaxation, unknotting, or decatenation. These reactions are mediated through a transient double-strand break whose 5'- extremities are covalently bound to tyrosine residues of the enzyme; a second DNA segment is captured and transferred through the break by conformational changes, and the DNA is religated. Religation is inhibited by agents which are widely and successfully employed in antitumour chemotherapy (etoposide, adriamycin, amsacrine). Chemotherapy is followed in a significant proportion of cases by the emergence of secondary leukemias, whose cells show characteristic chromosome translations which in some cases have been shown to occur at high-affinity binding sites for topoisomerase II. We and others have shown that these drugs induce translocations, sister chromatid exchanges, and mutations in cultured cells. These effects are likely to involve drug-induced errors during the religation reaction which result in illegitimate ligation to extraneous DNA ends, for example those bound to other topoisomerase II molecules or at DNA breaks. The possible significance in spontaneous mutation and chromosome rearrangement of the error-prone nature of this reaction merits examination. The underlying molecular processes have not been elucidated, and experimental approaches to investigate them will be discussed.
41 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
THE IMPACT OF DNA REPAIR ON CHEMICALLY-INDUCED GERM CELL MUTAGENESIS Madeleine Nivard, John Wijen. Ekkhart Vogel Leiden University, MGC, 2300 RA Leiden, The Netherlands.
The large class of alkylating agents (AAs) was and still is extensively studied with respect to their mutagenic action principles in germ cells of both mouse and Drosophila. The analytical tools available when using Drosophila concern both genetic and molecular assays. The genetic tests enable to quantify excision repair and clastogenic potency in relation to the functionality of the AA, its nucleophilic selectivity and its carcinogenic potency. For a more selected group of agents their molecular spectra has been studied. The combined studies obtained from post- meiotic male germ cells revealed so far five distinct classes of alkylating agents: 1. SNl-type monofunctional AAs; agents with low s value. Alkylation of oxygen positions in the DNA determines their high mutagenic and carcinogenic activity. Examples are ENU (A^-ethyl-Af-nitrosourea), MNU (N-methyl-JV-nitrosourea) and DMN (jV-nitrosodimethylamine); 2. SN2-type monofunctional AAs; agents with high s value which predominantly react with ring nitrogens in DNA. They are weakly mutagenic and carcinogenic probably because the nitrogen DNA adducts are very well repaired. Representatives of this class are MMS (methyl methanosulfonate) and EO (ethylene oxide); 3. Polyfunctional AAs, for instance melphalan, chlorambucil, and BCNU (l,3-bis(2-chloroethyl)-l-nitrosourea). Results suggest that the formation of DNA crosslinks is responsible for their high carcinogenic and mutagenic potency; 4. Agents able to form Etheno DNA-adducts (vinylchloride, vinylcarbamate); or 5. Bulky adducts (aflatoxin B|). From the first three classes of mutagens data are available from the specific locus test in the mouse, the only other germ cell system which has extensively been studied. Comparison of Drosophila results with genetic damage in male mice revealed a most unexpected but exciting outcome. In brief, all three classes of mutagens are highly active mutagens in postmeiotic germ-cell stages. In premeiotic stages only the ENU- type mutagens and the polyfunctional agents showed genotoxic activity. The mutagenic effects of the MMS-type mutagens are very much reduced in these premeiotic stages. This phenomenom seems to be related with DNA repair activity. A characteristic feature of post-meiotic male germ cells of both mouse and Drosophila is the absence of DNA repair enzymes for a period of, respectively, 14 and 3-4 days. The consequence is that in both the mouse and Drosophila spermatozoa and spermatid stages are susceptible to all kinds of DNA alkyl adducts leading to mutations. The relevant question to be addressed is if low TD50 values are related to mutagenic activities in a wide range of germ cells. Consideration of mutation spectra in postmeiotic male germ cells reveal remarkable similarities between mouse and Drosophila. Both MMS-type mutagens and cross- linking agents predominantly produce deletion mutations. This is quite in contrast to what is generally found in unicellular systems and in mammalian cells in culture.
42 Session III: DNA Repair in Eukaryotes and its Relation to Human Diseases
DNA DAMAGE IN LYMPHOCYTES AND AORTAL ENDOTHELIA OF DIABETIC AND CHOLESTEROL- FED RATS Rudolf Stetina1, Vera Stetinova2, Vojtech Grossmann2, Andrew R. C. Collins3 'inst. Exp. Med., Lab. Environ. Toxicol. Acad.Sci., Olesnice v O.h., 517 83, Czech Republic 2 Inst. Exp.Biopharm. Joint Labs of Czech Academy of Sciences and PRO.MED.CS Co., Heyrovskeho 1207, Hradec Kralove, 500 02, Czech Republic 3Rowett Res. Inst., Greenburn Road, Bucksburn, Aberdeen, AB2 9SB, Scotland, UK.
The DNA damage was followed in both sexes of diabetic and cholesterol-fed Wistar rats using the comet assay. Cholesterol was applied (2% of the standard diet intake) for the period of 1 (males) or 2 month (females), alloxan was given i.v. (40 mg/kg) to induce hyperglycaemia. DNA damage was measured in lymphocytes, endothelial cells isolated from aorta, and in liver cells isolated by brief trypsinisation of the liver tissue, After 1 month of cholesterol feeding much higher increase of single strand DNA breaks (SSB) in lymphocytes was found in males from (0.15 to 1.2 SSB /109 daltons) compared to females ( from 0.16 to 0.2). Similar increase was seen in endothelial cells of the aorta. When the DNA was incubated with endonuclease III before electrophoresis, a high increase of SSB was observed in lymphocytes of cholesterol-fed males (up to 2.6 SSB/109 daltons) while much lower increase was found in females ( up to 0.4 SSB). Similar differences between sexes were found in endothelial aortal cells. Similar results were obtained with hyperglycaemic rats after the application of alloxan. DNA damage in endothelial cells may represent an early injury to these cells by cholesterol (or its oxidation products). The oxidative damage in lymphocytes may be a very sensitive indicator of the damage in endothelia.
43 POSTERS
left BLANK PL9800379 Poster Session Poster N° 10
TRANSCRIPTIONAL INDUCTION OF THE SACCHAROMYCES CEREVISIAE MAGI GENE, IN RESPONSE TO DNA-DAMAGING AGENTS, REQUIRES NEITHER THE MEC1, POL2, RAD53 NOR DUN1 REGULATORY GENE PRODUCTS Malgorzata Adamczyk. Adrianna Nowicka and Ewa Sledziewska-Gojska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
The S. cerevisiae MAGI gene is encoding alkylation repair DNA glycosylase. Besides 3-methyladenine, 7-methylguanine and 7- methyladenine MAGI encoded glycosylase removes also ethenobases, induced in cellular DNA by vinyl halides, and DNA deamination product hypoxanthine. Transcription of the MAGI gene is induced by DNA damaging agents such as MMS, UV or NQO. Transcriptional activation of number of genes involved in DNA metabolism is a part of eukaryotic response to DNA damage. POL2-MECl-RAD53-DUNl-sign&\ transducing pathway has recently been postulated to be involved in the regulation of the general response of the S.cerevisiae cells to DNA damage. We have analysed the expression of MAGI in cycling S. cerevisiae strains carryng MAGlr.lacZ fusion and deficient in either POL2, MEC1, RAD53 or DUN1 function, p-galactosidase activity has been assayed in cells exposed to MMS or UV. It has been found that, in contrast to DNA-damage inducible RNR genes, neither mutation in the sensory C-terminal part of polymerase e (po!2-ll), nor inactivation of Mecl, Rad53 or Dunl cellular kinases (in mecl-, sadl-1 or dunl-21::TRPl, dunl-25::TRP, respectively) affects the induction of MAGlr.lacZ fusion in response to MMS or UV treatment, as compared to control MEC, POL2, RAD53, DUN1 strains. Thus expression of MAGI may be subjected to an alternative regulatory circuit, the components of which remain to be established.
47 Poster Session Poster N° 31
POSSIBLE INVOLVEMENT OF DNA REPAIR PROCESSES IN THE INDUCTION OF APOPTOSIS IN TUMOR CELLS TREATED WITH ANTITUMOR DNA CROSSLINKING AGENTS Ewa Augustin and Jerzy Konopa Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Gdansk, Poland.
Antitumor DNA crosslinking agents as the first biological effect induce the cell cycle arrest of tumor cells in the G2 phase of the cell cycle (rev. J. Konopa, Biochem. Pharmacol. 37, 2303, 988). We showed that tumor cells blocked in the G2 phase by such DNA crosslinking agents as anthracyclines, ledakrin, melphalan, mitomycin C, CCNU, chlorambucil undergo programmed cell death (apoptosis) (A. Skladanowski, J. Konopa, Biochem. Pharmacol. 46, 375, 1993; E. Augustin, A. Skladanowski, J. Konopa, unpublished data). One of the possible mechanisms of apoptosis could be related to induction of repair processes of damaged DNA due to the covalent binding to DNA of crosslinking agents, which results in induction of poly (ADP-ribose) synthesis and in consequence, in depletion of cellular NAD and ATP. Leukemia L1210 cells arrested in the G2 phase by mitomycin C or melphalan, at concentration corresponding to EC90, after 14 hrs of treatment begin undergo apoptosis which was traced both electrophoretically by internucleosomal DNA cleavage and by observation of morphological changes. In cells treated with the studied compounds we observed decline in the level of NAD to complete depletion after prolonged incubation time. This effect was followed by depletion of cellular ATP. These results confirm the hypothesis that DNA repair processes could be involved in the reduction of apoptosis by DNA damaging agents.
48 Poster Session PL9800380 Poster N° 34
ANALYSIS OF POLAR DNA ADDUCTS FORMED IN MOUSE EPIDERMIS FROM DIBENZ[A,J]ANTHRACENE Suryanarayana V. Vulimiri1, Wanda Baer-Dubowska2 and John DiGiovanni1 'UT MD Anderson Cancer Center, Smithville TX 78957, USA 2Department of Pharmaceutical Biochemistry K.Marcinkowski University of Medical Sciences, Poznari, Poland.
The formation of DNA adducts in mouse epidermis has been examined following topical application of dibenz[aj]anthracene (DB[aJ]A) and its derivatives. DNA adducts were analysed by 32P-postIabeling coupled with HPLC or TLC. The HPLC profiles of 3',5'- diphosphodeoxyribonucleotide adducts obtained from epidermal DNA isolated from mice treated with parent compound revealed 12 different adduct peaks. Five of these peaks were identified as derived from the reaction of anti- and syn-diol epoxides with dGuo and dAdo. Approximately half of the remaining adducts was represented by more polar adducts eluted prior to standard anti- and syn-diol epoxide DNA adduct peaks. In order to identify these adducts, polar metabolites of DB[aj]A, i.e. DB[aj]A-bis dihydrodiol, DB[aj]A 3,4,10-triol or DB[aj]A-3,4,ll-triol were applied to the shaved backs of mice and epidermal DNA was isolated 24 h later and 32P-postlabeled. DNA adducts formed by the DB[aj]A metabolites were analysed by TLC and compared with the adducts formed by parent compound. DB[aj]A produced again at least 15 DNA adduct spots in TLC chromatograms of mouse epidermal DNA including very polar adducts. However, analysis of DNA from triol treated mice did not show any adduct spots that corresponded to polar adducts formed by the parent compound. The bis - dihydrodiol yielded at least three weak adduct spots on the TLC chromatograms which were also present in the DNA from 3,4-diol treated epidermis and they corresponded to similar adduct from the parent compound. Thus some of the polar DNA adducts formed from DB[aj]A derived from further metabolism of the bay-region bis - dihydrodiol. However the bis-dihydrodiol did not possess skin tumor initiating activity at a dose of 400 nmol per mouse. Thus, the overall contibution of the bis-dihydrodiol-derived DNA adducts to the tumor initiating activity of DB[aJ]A appears to be relatively low.
49 PL9800381 Poster Session Poster N° 18
GENE SPECIFIC REPAIR OF UV-INDUCED CYCLOBUTANE PYRIMIDINE DIMERS IN RETROVIRUS-INFECTED HUMAN CD4+ LYMPHOCYTES Zdena Bartosova1'2, William Reinhold2, Martin Kolisek1, Miroslav Pirsel1, Maria Zajac-Kaye2, Vilhelm A. Bohr3, and Ivan D. Horak2'4 'Cancer Research Institute, SAS, 812 32 Bratislava, Slovakia, 2National Cancer Institute NIH, Bethesda, MD 20892, USA, 3National Institutes on Aging, NIH, Baltimore, MD 21224, USA, 4Janssen Research Foundation, Titusville, NJ 08560-0200, USA
HTLV-1 retrovirus associated Adult T-cell leukemia/lymphoma (ATLL) is recently uncurable cancer. As the molecular mechanism of disease development is not fully understood, we started to investigate the rate and the extent of DNA damage removal in HTLV-1 negative and positive cells. DNA repair was assessed on the gene specific level and the transcription rate of the genes was determined by in vitro nuclear run-on assay. Proto-oncogene c-myc was found being similarly expressed in proliferating fresh CD4+ lymphocytes, Jurkat T-cell line (HTLV-1 negative), Hut 102 T-cell line (HTLV-1 positive), and in the fresh ATLL patient cells. However, removal of UV-induced cyclobutane pyrimidine dimers (CPD) from this gene was signifcantly different between cell strains, except for fresh CD4+ cells and Jurkat T-cell line, where about 60% of CPD was removed within 24 h after UV (15 J/m2). Hut 102 T- cell line displayed somehow faster and higher repair of CPD in the c-myc gene than did non-infected cells. Interestingly, in fresh ATLL patient cells virtually no removal of damage occured. We explore further the possibility of DNA repair impairment as the factor contributing to ATLL development.
50 Poster Session Poster N° 20
ENDOGENOUS AND EXOGENOUS DNA LESIONS RECOGNIZED BY N-ALKYLPURINE-DNA GLYCOSYLASES Ewa Borvs and Jarostaw T. Kusmierek Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
The combined action of glycosylases and abasic site-specific endonucleases on damaged bases in DNA results in single strand breaks. In plasmid DNA, as a consequence, the covalently closed circular (ccc) form is converted to the open circular (oc) form and this can be quantitated by agarose gel electrophoresis. We studied DNA lesions sensitive to E. coli AlkA and cloned human ANPG-40 glycosylases which are known to excise alkylated bases and etheno adducts. To our surprise pBR322 and pAlklO plasmids not pretreated with mutagens, were cleaved by both glycosylases in the presence of enzymes possessing endonucleolytic activity (Nth, Fpg, Exo HI). The plasmids pretreated with chloroacetaldehyde (CAA), a mutagen forming etheno adducts, exhibited enhanced sensivity towards glycosylases tested. On the other hand, the plasmids pretreated with malondialdehyde (MDA), an endogenous DNA-damaging agent, were not cleaved to a higher extent than untreated controls. Our results indicate that plasmids isolated from bacteria contain unknown endogenously formed adducts recognized by ANPGs. This limits the exploitation of conformational changes of plasmid DNA to study the substrate specificity of N-alkylpurine-DNA glycosylases. Nevertheless, the method can be used to distinguish between different types of DNA adducts, e.g between etheno and adducts.
51 Poster Session Poster N° 22
INTRODUCTION OF THE E.coli nth GENE INTO THEpso3-l MUTANT OF S.cerevisiae: EFFECTS ON SENSITIVITY TO HYDROGEN PEROXIDE AND PARAQUAT Jela Brozmanova', Viera Vlckova2, Zaneta Mikulovska1, Ivana Fridrichova1, Eva Farkasova1, Richard P. Cunningham3 and Joao A.P. Henriques4 1 Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia, 2 Department of Genetics, Faculty of Sciences, Comenius University, 842 15 Bratislava, Slovakia 3 Department of Biological Sciences, SUNY at Albany, Albany, N.J. 12222, USA 4 Centra de Biotecnologia, Federal University de Rio Grande do Sul, 91501 Porto Alegre, Brasil
Reactive oxygen species can cause extensive damage to DNA. Unrepaired oxygen radical-derived DNA lesions have been implicated as a causative factor in both cancer and aging. Repair of most forms of oxidative DNA damage is mediated via a base excision repair pathway. One of the key components of the base excision-repair pathway in Escherichia coli is endonuclease III, the product of the nth gene. In Saccharomyces cerevisiae several endonuclease Ill-like proteins have been identified. Among them the endonuclease three-like glycosylasel and redoxyendonuclease seem to be most serious candidates for functional yeast analogs of the Nth protein. Recent studies suggest existence of family of genes participating in the repair of oxygen radical- induced DNA damage in yeast. S.cerevisiae pso3-l allele confers sensitivity to treatment with photoactivated mono- and bi-functional psoralens, but also to hydrogen peroxide, paraquat and formaldehyde, suggesting that pso3-l is also impaired in mechanism(s) of the repair of oxidative stress-related DNA lesions. To contribute to understanding of a possible role of the PSO3 gene in the repair of oxygen radical-derived DNA lesions we have used the E.coli nth gene as a probe for complementation of its sensitivity to hydrogen peroxide and paraquat. We have introduced the E.coli nth gene into the pso3-l mutant and the isogenic wild type cells. Nth protein was expressed in both strains used. Nth protein was able significantly reduce the toxic effects of both hydrogen peroxide and paraquat in the pso3-l mutant. The present results indicate that the toxic DNA lesions induced by oxidative stress can be repaired in yeast by the E.coli nth gene product.
52 Poster Session Poster N° 9
MNNG-INDUCED RecBCD- ENZYME DEPENDENT DNA DEGRADATION IN recA13 MUTANT CELLS IS NOT A CAUSE OF THEIR HIGH SENSITIVITY TO THIS AGENT Miroslav Chovanec1. Dana Vlasakova1, Geoff P. Margison2, Maria Naslund3 and Jela Brozmanova1 1 Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia, 2 CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester M20 9BX, UK 3 Department of RaRadiobiologyd , Stockholm University, S-106 91 Stockholm, Sweden
RecA mutations are remarkably pleiotropic, affecting also the response of bacterial cells to alkylation damage. We were interested whether high sensitivity of the recA mutant to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) might be caused by the changed level of DNA repair protein, O6-methylguanine-DNA methyltransferase (MTase), either at its constitutive or induced state. We have measured the level of MTase in the adapted and non-adapted recA13 mutant and wild type cells. We have found approximately two-fold lower constitutive level of MTase in the recA13 mutant. However, since the adaptation-associated increase in MTase in the recA13 genetic background was comparable to that observed in wild type cells, we suggest that the adaptive response is not dependent upon a functional RecA protein. The adapted recA13 strain nevertheless was vastly more sensitive to MNNG than non-adapted wild type strain. We therefore assumed that the extreme sensitivity of the recA13 mutant to MNNG is probably caused by some nonspecific effect on recovery such as "reckless" degradation of DNA. Indeed, the exposure to MNNG resulted in a substantial degradation of cellular DNA in recA13, but not in the wild type cells. On the other hand, we have found no DNA degradation in the recA13 cells lacking the RecBCD enzyme, exonucleae V. It shows that DNA degradation in the recA13 background after MNNG treatment is due to the nuclease activity of the RecBCD enzyme. Since the recA13 recB21 double mutant is much more sensitive to MNNG than the recA13 single mutant we conclude that the MNNG- induced RecBCD-dependent DNA breakdown in the recA13 background is not a prime cause of their hypersensitiviry to this agent.
53 Poster Session Poster N° 23
ALTERNATIVE APROACHES TO THE MEASUREMENT OF REPAIR OF OXIDATIVE DNA DAMAGE IN HUMAN CELLS. Maria Dusinska1, Martina Somorovska1, Helena Petrovska', Rudolf Stetina2 and Andrew Collins3 'institute of Prevetive and Clinical Medicine, Bratislava, Slovakia Institute of Experimental Medicine, Czech Academy of Sciences, Olesnice, Czech Republic Rowett Research Institute, Bucksburn, UK
A common approach to the measurement of DNA repair is to follow the removal of lesions. Strand breaks and oxidised bases can be detected with the alkaline comet assay (single cell gel electrophoresis) in combination with lesion-specific endonucleases. We have monitored in vivo recovery after H2O2 treatment of lymphocytes from patients with diabetes or ankylosing spondylitis (AS), compared with normal subjects. Normal and diabetic subjects show a range of responses in terms of capacity to remove damages; AS patients are uniformly poor at recovery. Whether this is because of depressed repair or low antioxidant status is currently under investigation. In a novel modification of the comet assay, we use nucleoid DNA from specifically damaged cells, in the gel, as substrate for repair enzymes in a simply prepared extract from lymphocytes. We have demonstrated, in extracts from several individuals, significant activity against oxidative damage. This assay is being developed as a biomonitoring tool, and will be used to investigate the cause of variability between individuals in rates of recovery from oxidative damage. This work is supported by EC contract CIPA-CT94-0129
54 Poster Session PL9800382 Poster N° 12
INACTIVATION OF A PROTEIN KINASE ENCODED BY DUN1, THAT CONTROLS THE DNA DAMAGE RESPONSE IN SACCHAROMYCES CEREVISIAE, RESULTS IN A HIGH FREQUENCY OF rho" MUTANTS Marta U. Fikus, Joanna Rytka and Zygmunt Ciesla Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
In budding yeast, several genes have been identified that control the ability of cells to activate the trancriptional response to DNA damage. DUN] encodes a protein kinase that is activated by treatment of cells with DNA-damaging agents and is involved in induction of several genes, e.g. RNR1, RNR2, RNR3 and DIN7. The dunl mutants have been shown to be more sensitive to UV-light or MMS (methyl methansulfonate) as compared to the parental DUN1 strains. We have found that inactivation of DUN1, by transposon insertion, results in a high frequency of mitochondrial respiratory deficient mutants. This phenotype of dunl is complemented by plasmid pZZ48 carrying wild- type DUN1, but not by pZZ82 or pZZ99 encoding inactive forms of the Dunl kinase. Both the genetic analysis and DAPI staining of DNA indicate that respiratory deficient cells, arising in the dunl background, predominantly represent rho" and not rho0 mutants. This seems to be an interesting observation in light of a well known fact that large deletions of mitochondrial DNA (mtDNA) are responsible for the phenotype of rho' mutants. However, as segments of the remaining mtDNA can be amplified many times, the total quantity of mtDNA is the same in rho' and rho+ strains. Experiments designed to examine whether the dunl deficiency results specifically in the instability of the mitochodrial genome or perhaps, it may also affect general stability of the yeast DNA, are in progress.
55 Poster Session Poster N° 35
USE OF REPAIR ENDONUCLEASES FOR CHARACTERIZATION OF DNA DAMAGE INDUCED BY N-HETEROCYCLIC AROMATIC HYDROCARBONS Gabelova, A.', Bacova, G.', Slamenova, D.', Perin, F.2 'Cancer Research Institute, Bratislava, Slovak Republic " Institute Curie, Orsay, France
Both 7H-dizbenzo[c,g]carbazole (DBC) and N-methyldibenzo[c,g] carbazole (MeDBC) belong to the group of N-heterocyclic aromatic hydrocarbons (NHA). As many other chemical carcinogens, DBC and MeDBC require metabolic activation to electrophilic species before they can interact with DNA and other macromolecules and exert their mutagenic and carcinogenic effects. The microsomal cytochromes P450 catalyze the utilization of oxygen and in so doing generate potentially toxic reactive intermediates which bind covalently to DNA, forming DNA-adducts. Two ways of biotransformation of DBC are suggested: at its ring-carbon atoms, as with PAH, and also at nitrogen position. It is supposed that the N-7 position plays an important role in liver carcinogenicity. This suggestion is supported by the fact that, MeDBC, which is the N-methyl derivative of DBC, lacks hepatocarcinogenetic potential. Different ways of metabolic activation were used for DBC and MeDBC: i) subcellular S9 fraction; ii) V79 cell lines with stable expression of cDNA of several cytochromes P450; iii) human hepatic cell line Hep G2. In order to characterize DNA damage induced by DBC and its methyl derivative MeDBC, a set of repair endonucleases, which specifically recognize certain DNA base modifications and sites of base loss (AP sites) was used. For detection of DNA damage induced by DBC and MeDBC a modified alkaline single cell gel electrophoresis has been applied.
56 Poster Session Poster N° 1
THE ASSYMETRY OF MUTAGENESIS DURING REPLICATION OF LEADING AND LAGGING DNA STRANDS IN E. COLI CHROMOSOME Damian Gawel1. Piotr Jonczyk1, Roel M. Schaaper2 and Iwona J. Fijalkowska1 'institute of Biochemistry and Biophysics, PAS 02-106 Warsaw, Poland; National Institute of Environmental Health Sciences, Research Triangle Park, NC USA
Mutations in DNA may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying a DNA template strand. It is important to understand the factors which contribute to the accuracy of this process. We investigated the mutagenic potentials of leading and lagging strands DNA replication in the model organism Escherichia coli. In contrast to the previously described assays based on plasmid replication or measurements of fidelity using in vitro mutagenesis assays, we present a novel approach to a such key question which allow us to measure the differences in the fidelity of synthesis of two strands during chromosomal replication in vivo. We constructed 4 pairs of strains differing in the orientation of a mutational target, the lac operon, with respect to the origin of chromosomal DNA replication. In one strain of each pair, a defined sequence is replicated as a leading strand, while in the parallel strain, the same sequence is replicated as a lagging strand. To measure the specificity and frequency of mutagenesis we used a set of lacZ alleles constructed in J. Miller's laboratory. These alleles represent different frameshift mutations within lacZ gene. Each frameshift mutation can be reverted to Lac+ by single, one nucleotide, specific frameshift event. Our results, obtained with mutator strains impaired in mismatch repair activity, clearly indicate that there is a difference in the fidelity of replication between the two replicating DNA strands. The differences in the level of mutagenesis between the two strands were observed for -1G and +1G but not for -1A and +1A frameshifts.
57 PL9800383 Poster Session Poster N° 21
INHIBITION OF DNA SYNTHESIS ON TEMPLATES OXIDIZED BY HYDROXYL RADICAL GENERATING SYSTEM M.A.Graziewicz', T.H.Zastawny2, R.Olinski2, J.Siedlecki3, B.Tudek1 'institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland, department of Clinical Biochemistry, Medical School, Bydgoszcz, Poland, institute of Oncology, Warsaw, Poland
Hydroxyl radicals when interacting with DNA produce many different lesions, among which 8-hydroxy- and imidazole ring-opened purines are quantitatively prevalent. To study the effect of base damage on DNA replication, M13mpl8 phage DNA was oxidized in vitro by hydroxyl radical generating system -hypoxanthine/xanthine oxidase/Fe+3/EDTA. The closed- circular form of the phage DNA was isolated and used as a template for DNA synthesis by 3 different DNA polymerases: T7, DNA polymerase, Klenow fragment of DNA polymerase 1 from E.coli and DNA polymerase (J from calf thymus. The reaction products were analysed by polyacrylamide gel electrophoresis and inhibition of DNA synthesis was assessed by scanning of the intensity of stop bands opposite damaged bases on autoradiograms. Inhibition of DNA synthesis occurred with different intensity and in a sequence dependent manner. For prokaryotic polymerases (T7, Klenow) oxidized pyrimidines were the major stop sites (100% and 90% of the maximal intensity of stop bands opposite Thy and Cyt, respectively, versus 40% and 20% opposite Ade and Gua). For DNA polymerase (3 oxidized purines were the major stop sites (45% and 42% of the maximal intensity of stop bands opposite Thy and Cyt, respectively, versus 85% and 55% for Ade and Gua). (5 polymerase showed a tendency to stop replication opposite purine adjacent to pyrimidine, while polymerase T7 and Klenow fragment bypassed more easily the same sequence. For all 3 polymerases oxidized purines stopped replication when present in the middle of purine tract containing Ade and Gua. Gas chromatography-mass spectrometry (GC- MS) analysis of oxidized templates has shown that hydroxyl radical- mediated oxidation of this ssDNA produced mainly Fapy-Gua (2,8 nmoles/mg DNA, 30-fold increase), 8OH-Gua (1.4 nmoles/mg DNA, 1.6- fold increase), Fapy-Ade (0.85 nmoles/mg DNA, 2.5-fold increase) and thymine glycol (0.74 nmoles/mg DNA, 4.3-fold increase). These results suggest that imidazole ring-opened purines are potentially lethal lesions but their ability to block DNA synthesis depends on the surrounding nucleotide sequence and on the interaction with DNA polymerase active site.
58 Poster Session Poster N° 36
DNA REPAIR OF ADRIAMYCIN INDUCED DAMAGE IN MOUSE AND HUMAN CELLS DIFFER IN CATALASE ACTIVITY Beata Gruber, Elzbieta Anuszewska. Drug Institute, Chelmska Str. 30/34, 00-725 Warsaw, Poland.
Adriamycin, an anthracycline antibiotic is one of the most effective anticancer agent but chemotherapy is severely limited by the occurrence of cytotoxicity. Mechanism of adriamycin cytotoxicity appears to be linked to the formation semiquinone free radical. In the presence of molecular oxygen, the semiquinone rapidly reduces the oxygen to superoxide that is converted to hydrogen peroxide. Catalase is a major antioxidant enzyme involved in the detoxification of hydrogen peroxide. Thus, we have expected that the cells may differ in catalase activity and when treated with adriamycin may give diversified answer in regard to DNA damage and repair. On the basis of determination of catalase activity in several human cell lines and primary cultures of mouse embryo cells, the following cells with different catalase activity were chosen for further studies: normal human cells ATCC no. CRL-2088 (CAT 72.4 i.u./mg protein); xeroderma pigmentosum cells ATCC no. CRL-1307 (CAT 20.6 i.u./mg protein); mouse embryo fibroblasts derived from AKR strain (CAT 71.3 i.u./mg protein) and BALB/c strain (CAT 32.0 i.u./mg protein). DNA damage and repair in these cells following exposure to various concentrations of adriamycin were studied by alkaline elution method at 0, 24 and 48 hrs after treatment. Adriamycin (0.5 u,g/ml) induced single strand breaks in CRL-2088 cells were in part repaired within 24 h after drug removal and completely repaired within 48 h. In AKR cells breaks were incompletely repaired till 48 h after treatment with adriamycin at the same concentration. No repair of single strand breaks induced by adriamycin was observed in CRL- 1307 and BALB/c cells up to 48 h.
59 Poster Session Poster N° 4
IS THE DNA POLYMERASE III INVOLVED IN THE PREFERENTIAL TRANSCRIPTION COUPLED DNA REPAIR? Elzbieta Grzesiuk, Agnieszka Gozdek, Anetta Nowosielska, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
We have observed decline in mutation frequency under conditions of transient amino acids starvation (MFD phenomenon) for MMS induced E. coli argE3oc strain. Arg+ revertants in argE3oc are the result of z//wwZ)C-dependent, supL suppressor formation arising by AT to TA transversion. We have previously shown that in E. coli strain mutated in e subunit of DNA polymerase III (E. coli dnaQ49) MMS induced argE3—>Arg+ reversions are not subjected to MFD. We have found that using E. coli dnaQ49 strain transformed with plasmids harbouring combinations of the umuD(D')C genes, repair of MMS induced lesions depends on the availability of UmuD 'Cl UmuD' proteins. There can be at least two explanations for these observations: (i) the kinetics of repair process is different in dnaQ' and dnaQ~ strains. (ii) e subunit of DNA pol III encoded by dnaQ gene is directly involved in MFD phenomenon. Here we try to check the second hypothesis. The phenotype of E. coli dnaQ49 is fully expressed at 37°C and at this temperature we do not observe decline in the frequency of MMS induced argE3—>Arg reversions. However, at permissive temperature of 30°C MFD phenomenon has been observed. This result suggests involvement of e subunit of DNA pol III in MFD. The same type of experiments, but with UV as mutagen are in progress.
60 Poster Session Poster N° 13
INFLUENCE OF NUCLEAR DNA POLYMERASES AND MSH3 GENE ON ADAPTIVE MUTATIONS IN YEAST SACCHAROMYCES CEREVISIAE Agnieszka Haias. Hanna Baranowska, Zofia Policiriska and Witold Jachymczyk Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Selection induced mutations that occur as specific and direct responses to environmental chal leges under intense prolonged selection in nondividing cells was discovered in yeast by Hall (1) and by Steele and Jinks-Robertson (2). We found previously that temperature sensitive cdc2-l mutation in DNA polymerase 5 strongly enhances the frequency of adaptive mutations in the yeast cells (3). Here we investigated the influence of mutation in other DNA polymerase genes and in MSH3 gene responsible for mismatch DNA repair process on the selective induced mutations at the LYS2 locus. We found that thermosensitive mutation in POL2 gene encoding DNA polymerase e increased ratio of adaptive mutation frequencies in similar way as it was found earlier for DNA polymerase 8. Also similar effect was observed in strains with deletion of MSH3 gene. Mutations in other DNA polymerases, including the essential DNA polymerase a and nonessential DNA polymerases B and Rev3p showed no effect on this process. The obtained results suggest that errors formed in DNA during elongation and their persistence in mutants deficient in post-synthetic mismatch repair activity seems to be the cause of the adaptive mutation appearence in the yeast cells.
1 .Hall BG (1992) Selection induced mutations occur in yeast. Proc.Natl. Acad. Sci.USA89:4300-4303 2.Steele BF, Jinks-Robertson S. (1992) An examination of adaptive reversion in Saccharomyces cerevisiae. Genetics 132:9-21 3. Baranowska H., Policiriska Z., Jachymczyk WJ (1995) Effects of theCDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae. Curr Genet 28:521-525
61 Poster Session Poster N° 39
EFFECT OF VITAMIN C ON BENZO(a)PYRENE-INDUCED DNA DAMAGE STUDIED BY COMET ASSAY Marzena Gajecka, Pawel Jaloszynski, Maciej Kujawski, Krzysztof Szyfter Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60 479 Poznan, Poland,
Peripheral blood lymphocytes (PBL) isolated from 19 healthy, female volunteers (age: 23-24, non-smoking) were exposed on ice to benzo(a)pyrene alone or together with vitamin C. DNA damage was measured by alkaline comet assay. In preliminary experiments dose- dependent curves were established showing a direct proportional response between 0.2 and 1 or 2 u,M of B(a)P and a clear saturation from 1-2 |LLM (up to 10 |J.M, higher concentrations were not been studied). B(a)P at cone. 1 \xM was used to analyse a potential protective effect of vitamin C. The latter concentration showed a significant increase in DNA migration (measured as comet length) in PBL and treated cells were able to repair such a damage within 25 min of incubation in B(a)P- free RPMI 1640 medium at 37°C. Vitamin C was applied at 40 and 100 U.M - the first cone, has been reported in literature as a mean level of vitamin C in human blood plasma. Both concentrations of vitamin C showed a clear protective effect when applied simultaneously with B(a)P, DNA migration was significantly decreased (as showed with Mann-Whitney U-test). Vitamin C also decreased the background level of DNA damage observed in negative control. This damage might be a result of the procedure and the effect of an antioxidant such as vitamin C indicates an oxidative nature of such DNA breaks. The effect of vitamin C on B(a)P-induced DNA damage supports a hypothesis assuming a contribution of oxidation process in formation of B(c/)P-induced DNA breaks.
62 Poster Session Poster N° 3
IN VIVO PROTEIN INTERACTIONS WITHIN THE E. COLI DNA POLYMERASE III CORE. Piotr Jonczvk1, AdriannaNowicka1, Iwona Fijatkowska1, Roel M. Schaaper2 and Zygmunt Ciesla1. 'institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; 2National Institute of Environmental Health Sciences, Research Triangle Park, NC USA
The mechanisms that control the fidelity of DNA replication are a matter of considerable interest. These mechanisms are being investigated by a number of approaches, including detailed structural and kinetic studies. Important tools in these studies are mutant versions of DNA polymerases that affect their activities, including fidelity of DNA replication. It has been suggested that the proper interactions within the core of DNA polymerase III (pol III) of E. coli could be essential for maintaining optimal fidelity of DNA replication [Maki H, Kornberg A (1987) Proc Natl Acad Sci USA 84: 4389-4392]. We have been particularly interested in elucidating physiological role of interactions between DnaE (a subunit - possessing DNA polymerase activity) and DnaQ (e subunit - possessing 3' -5' exonuclease proofreading activity) proteins. In attempt to achieve this goal we have used the Saccharomyces cerevisiae two-hybrid system designed to study specific interactions of proteins in vivo. In this report we demonstrate interactions between wild-type DnaE and DnaQ proteins as well as between DnaQ and HolE (6 subunit) proteins. We also have tested interactions of wild type DnaE and HolE proteins with three well known mutant forms of DnaQ, which lead to strong mutator phenotypes: MutD5, DnaQ926 and DnaQ49. Our results show that mutD5 and dnaQ926 mutations do not affect the e - a and e - 6 interactions. In contrast, the dnaQ49 mutation greatly reduces the strenght of interaction of the e subunit both with the a and 6 subunits. Thus, the mutator phenotype of dnaQ49 could be the result of an altered protein conformation of the e protein and in consequence, in its altered interactions within pol III core.
63 Poster Session Poster N° 16
PROTEIN FAMILY WITH POLY(ADP-RIBOSE)-BINDING SEQUENCE MOTIFS Hanna Kleczkowska1'2, Jutta M. Pleshke2 and Felix R. Althaus2 'institute of Nuclear Chemistry and Technology, Warsaw, Poland institute of Pharmacology and Toxicology, Zurich University, 8057 Zurich, Switzerland
PoIy(ADP-ribose) is synthesized in cells following induction of DNA damage. During DNA excision repair the processing of ADP-ribose is stimulated several thousand-fold. ADP-ribose polymers bind noncovalently to a specific group of proteins. The cycle of ADP-ribose polymer synthesis and degradation may be involved in a temporary dissociation from and reassociation of proteins with DNA. Moreover, activation of poly(ADP-ribose) synthesis by DNA strand breaks could represent a downstream signal leading to damage repair or apoptosis. Our goal was to identify the amino acid sequence motifs involved in noncovalent poly(ADP-ribose)-binding domains of histones [Panzeter et al. (93) J. Biol. Chem. 268, 17662]. We defined a tentative motif containing clusters of positively charged aa (R, K, H). Following this prediction we have used the 25 aa PSD peptide of MARCKS protein to test binding in the polymer blot assay. Targeted mutations in several aa of this polypeptide further refined the sequence requirements of the polymer-binding motif. Besides positively charged aa, a skeleton of hydrophobic aa (F, Y, M, L, L, I, V) turned out to be the essential of the motif, while polar aa (S, T, Q, N, E, D) were found to support the polymer binding. With the refined motif, we discovered a number of poly(ADP-ribose)- binding proteins which play an important role in DNA damage recognition and signaling: p53, p21, DNA-PK, XPA and MSH6. The polymer-binding sequences can be modeled as amphipathic a-helices, with defined hydrophobic-hydrophilic polarity of the peptide wheels.
64 Poster Session PL9800384 Poster N° 29
ANTIOXIDANT VITAMINS AS MODULATORS OF DNA DAMAGE AND REPAIR Maria Konopacka, Joanna Rzeszowska-Wolny Department of Experimental and Clinical Radiobiology, Institute of Oncology, Gliwice, Poland.
Ionizing radiation generates free oxygen radicals and ions which introduce damages to DNA and other cellular macromolecules. The level of DNA damage can be modulated by treating animals (or cultured cells) with antioxidant vitamins before or shortly after irradiation. We studied the effect of vitamins C, E and beta carotene on the level of DNA damage after irradiating animals or cultured cells with gamma rays (2 Gy, single dose). The effect of vitamin E and beta carotene was proportional to the dose; with increasing dose the observed post- irradiation DNA damage became lower. Vitamin C showed more differentiated effect. Mice treated with 400 mg/kg dose of vitamin C for 5 days before irradiation with gamma rays (2Gy, single dose) showed elevated level of DNA damage in some tissues, compared to irradiated and untreated group. Mixture of vitamins C, E and beta carotene added to culture medium of irradiated cells was able to increase the rate of DNA repair, as measured by the comet assay method.
65 PL9800385 Poster Session Poster N° 38
THE EFFECT OF SOME PESTICIDES ON CELL PROLIFERATION AND ABNORMAL MITOSES IN RAT LIVER G. Kostka1, D. Palut', J. Kopec-Szlezak2 'Department of Environmental Toxicology, National Institute of Hygiene, Warsaw, Poland 2 Department of Physiopathology, Institute of Haematology and Blood Transfusion, Warsaw, Poland
Some organohalogen pesticides have been classified as rodent non- genotoxic hepatocarcinogens and/or liver tumor promoters. The early hepatic changes produced by this type of carcinogens have in common an association with cell proliferation proposed to be the useful endpoint in screening of non-genotoxic, potentially carcinogenic substances. We studied the effect of organohalogen pesticides, bromopropylate [isopropyl 4,4-dibromobenzylate] and DDT [1,1-(2,2,2- trichloroethylidene)-bis-(4-chlorobenzene)] on DNA synthesis, mitotic activity including histological changes in rat liver. Males Wistar rats (Pzh:Wis) weighing 200±10g were used. Bromopropylate and DDT were administered orally either in a single or in repeated doses (given at 24h intervales) of 125, 250, 500mg/kg b.w. and 12 or 24mg/kg b.w. respectively. DNA synthesis was measured by [3H]thymidine incorporation into nuclear DNA. The number of mitoses was counted in 2000 hepatocytes and expressed per 1000 hepatocytes. Histological hepatic changes were determined by microscopy in preparations stained with haematoxylin and eosin. In the case of both pesticides, DNA synthesis and mitotic activity increased significantly in a dose-dependent manner after single administration of compounds. After prolonged administration of bromopropylate and DDT, DNA synthesis and number of mitotic figures tended to decline, though differences between two higher doses of bromopropylate (500 and 250mg/kg b.w.) and control animals were still significant. Histological and cytological analysis showed the presence of dose-dependent abnormal mitotic Figures (and c-mitoses) in hepatocytes of the bromopropylate and DDT treated rats. In particular after administration of higher doses of compouds, mitotic spindle exibited disturbances. Therefore, it is possible that both pesticides selectively affect functions of the mitotic spindle resulting in abnormal mitosis.
66 Poster Session PL9800386 Poster N° 14 REPAIR OF ALKYLATION DNA DAMAGES IN BY-2 TOBACCO CELLS Elzbieta Kraszewska. Marta Dobrzafiska, Barbara Tudek Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
We have investigated the response of BY-2 tobacco cells to different doses of the alkylating agent MMS (methyl methanesulphonate). Similarly to other eukaryotic cells, the treatment of BY-2 line with 1.2 mM MMS caused a substantial decreese in the cell number (29% alive) as compared to the survival of the cells after the treatment with 0.8 mM MMS (72% alive). Incubation of plant cell extract with [^H]MNU - methylated DNA substrate led to the release of 3-methyladenine and 7-methylguanine from DNA, as identified by HPLC chromatography. This suggests that the N-methylpurine DNA glycosylase activity is present in non-treated tobacco cells. Experiments characterizing this enzymatic activity are undertaken.
67 Poster Session Poster N° 25
SPONTANEOUS HOMOLOGOUS RECOMBINATION IN CHO CELL LINES DEFECTIVE IN DNA DAMAGE PROCESSING M. Kruszewski', H. Kruszewska2, N. Jarocewicz', H. Inaba3, P. Jeggo4, I. Szumiel1. 'institute of Nuclear Chemistry and Technology, Warsaw, Poland, 2Pharamaceutical Institute, Warsaw, Poland, 'National Institute of Radiological Sciences, Chiba, Japan, 4MRC, CMU, Univ. of Sussex, Brighton, UK
DNA spontaneous homologous recombination was studied using a DNA construct bearing tandem copies of LacZ marker gene containing a non- overlapping deletion. Four cell lines were used in this study: xrs6 defective in dsb (double strand break) repair; Pa 13 and Pb4 cells, probably defective in DNA crosslink repair; parental CH0-K1 cell line. After electroporation, stable transfectants containing a single copy of plasmid intergrated into the genome were isolated and the rate of spontaneous homologous recombination between the two copies of LacZ gene was estimated. The rate of spontaneous recombination in CHO-K1 cells was 1.9 event/locus/cell generation/105. The rate of spontaneous recombination in Pal3 was about 10-fold higher than in wt cells, on average 23.0 events/locus/cell generation/105. However, in Pb4 cells it was similar to that in wt cells, on average 3.0 events/locus/cell generation/105. In xrs6 cells we found a very high rate of spontaneous recombination, on average 600.0 events/locus/cell generation/103, i.e. 300 times higher than in wt cells. Our results confirm the role of dsb in initiation of homologous recombination. It seems also that homologous recombination does not play any role in processing of DNA crosslinks. This work was supported in part by EC grant CEC ERJBCIPD 930417 (PECO ACTION) and Japanese Science and Technology Agency fellowship (MK).
68 Poster Session Poster N° 24
HYDROGEN PEROXIDE-INDUCED DNA DAMAGE IN TWO L5178Y SUBLINES Marcin Kruszewski, Teresa Iwanenko, Maryla Wojewodzka Institute of Nuclear Chemistry and Technology, Warsaw, Poland
Two L5178Y (LY) mouse lymphoma sublines are cross sensitive to ionizing radiation and hydrogen peroxide. LY-S subline is 2 times more sensitive to ionizing radiation than LY-R subline, whereas LY-R subline is 3.6 times and 11 times more sensitive than LY-S subline to hydrogen peroxide at 37°C and 4°C, respectively. We used the alkaline version of comet assay combined with DNA oxidative lesion-specific enzymes to asses the hydrogen peroxide-induced DNA damage in LY cells. The amount of DNA breaks in hydrogen peroxide treated cells (25jiM 37°C lh) was 1.9 times higher in LY-R than in LY-S cells. In hydrogen peroxide treated LY-R cells we also found 5.4 higher amount of FPG- sensitive sites than in LY-S cells. In hydrogen peroxide treated LY-S cells we found 3.3 times and 1.4 times more EndoIII- and FPG-sensitive sites than in control cells, respectively. In hydrogen peroxide treated LY-R cells we found 2.8 times and 2.3 times more EndoIII- and FPG-sensitive sites than in control cells, respectively. The amount of DNA damage was in good correlation with cell sensitivity to hydrogen peroxide. This may indicate a relationship between DNA damage induction and cell sensitivity.
69 PL9800387 Poster Session Poster N° 11
DIN7, A DNA DAMAGE-INDUCIBLE GENE OF SACCHAROMYCES CEREVISIAE, IS A CLOSE HOMOLOG OF THE EXOl GENE INVOLVED IN DNA MISMATCH REPAIR Piotr Mieczkowski, Marta Fikus, Piotr Koprowski and Zygmunt Ciesla Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
A number of DNA damage-inducible (DIN) genes have been identified in Saccharomyces cerevisiae. They include genes involved in DNA synthesis or DNA repair. In this report we characterize a novel DNA damage-inducible gene, DIN7, the expression of which is elevated after exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydroxyurea). Expression of DIN7 is also induced during meiosis. By comparison of the predicted Din7 amino acid sequence to those in databases we found that it belongs to a family of proteins which include: S. cerevisiae Rad2 and its S. pombe and human homologs Radl3 and Xpgc; S. cerevisiae Rad27 and its S. pombe homolog Rad2. The greatest sequence similarity is found between Din7 and the product of the EXOl gene of S. cerevisiae, which is endowed with 5'-3' exonuclease activity. It has been recently shown that Exol is involved in DNA mismatch repair and is required for recombination of direct repeats. In order to verify the hypothesis that Din7 is a functional homolog of Exol, we have constructed the pPKl plasmid which carries DIN7 fused to the GAL10-CYC1 promoter. The plasmid has been introduced into different S. cerevisiae exol strains designed to study mutability and various types of recombination. Results of the tests aimed at examination, whether overexpression of Din7 can complement the mutator phenotype of the exol strains and their recombination deficiency, will be presented.
70 Poster Session PL9800388 Poster N° 30
REPAIR OF DNA DAMAGE AFTER IRRADIATION WITH VARIOUS TYPE OF RADIATION W. Niedzwiedz, A. Cebulska-Wasilewska Radiation and Environmental Biology Department, Institute of Nuclear Physics, Krakow, Poland
We have applied the single cell gel electrophoresis technique (SCGE) also known as the Comet assay to study kinetics of the DNA damage repair after exposure to different types of radiation. High LET neutrons from 252Cf source, and low LET y-rays from 60Co were chosen for those studies. Lymphocytes isolated from the whole blood (suspended in RPMI 1640 with a supplement of 15% calf serum) were irradiated with various doses of gamma radiation or neutrons. Following irradiation kinetic of repair in 37°C was investigated by an estimate of residual DNA damage after various times of post-irradiation incubation (5, 10, 15, 30 and 60 min). Percent of the DNA in the comet tail and DNA tail moment were measures used for an evaluation of the DNA damage. In case of y- irradiation within first 15 min of post-exposure incubation the tail moment decreased to about 70% of initial damage, after following 15 min most of the damage was repaired. After irradiation with neutrons within first 30 min the tail moment decreased to about 50%. At one hour after irradiation 20% of residual DNA damage was still observed. The level of unrepaired DNA damage could represent the double strand breakes, which need a longer time for rejoining.
71 Poster Session Poster N° 41
DNA SINGLE STRAND BREAKS AND DNA REPAIR IN THE LYMPHOCYTES OF WOODEN FURNITURE WORKERS Jadwiga Palus, Elzbieta Dziubahowska, Konrad Rydzynski Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Lodz, Poland
Several studies reported mutagenicity or genotoxicity of the extracts of certain woods. So far there is only one study assessing the genotoxic effects in the woodworkers, which indicated an increase of the frequency of chromatid breaks in the pheripheral lymphocytes of workers exposed to fumes emitted from heated woods in a Finish factory. Most of them worked in small factories employing less than 100 persons. The data for Poland indicate that about 100,000 workers in wood and furniture industries are exposed to wood dust and the wood dust concentration may range from 0,1 to 83,00 mg/m3, depending on the type and volume of manufacture. DNA single-strand breaks (sbs) in peripheral lymphocytes of wooden furniture workers were detected by microfiltration method and compared to that of smoking and non-smoking control persons. Cigarette smoking caused a small and statistically not significant increase in sbs incontrol persons (6%). Statistically signifcant increases in the number of DNA single strand breaks were detected in smoking workers compared to non- smoking workers (16%) and smoking controls (15%). DNA repair in the lymphocytes of wooden furniture workers was also assessed. The level of the DNA repair increased 5 fold in smoking workers and 6 fold in nonsmoking workers compared to respective controls. Cigarette smoking caused only a small increases in sbs in control persons (1,8 times). We suggest that the occupational exposure to wood dust and the substances emitted the coating materials of the furniture may represent a highly significant risk in carcinogenesis.
72 Poster Session PL9800389 Poster N° 40
EXPRESSION OF ATM GENE IN VITRO IN LYMPHOCYTES OF PATIENTS WITH ATAXIA TELANGIECTASIA AND OF THEIR PARENTS Andrzej L. Pawlak1, Agata Filip 2, Danuta Rozynkowa 2, Tomasz Kmiec
Jacek Pilch 4 1 Institute of Human Genetics, Pol. Acad.Sci., Poznari, Poland 2 Department of Human Genet., Medical University, Lublin, Poland 3 Center of Child Health, Warsaw, Poland 4 Il-nd Chair and Clinics of Pediatrics, Silesian Medical University, Katowice, Poland
Increased sensitivity to ionizing radiation of ATM gene carriers in comparison to control persons remains a diagnostic challenge. The increase in number of chromatid breaks in caffeine-treated cultures of ATM heterozygotes may be considered, both, as a possible diagnostic approach and an interesting aspect of ATM-heterozygous cells behavior in vitro (Mutat. Res., 230, 197-204, 1990). Presumably, in presence of caffeine, as a result of release of the damage induced cell cycle block, the damaged cells enter mitosis and display aberrations. We have shown that in vitro challenge of human mitogen stimulated lymphocytes with UVB (40 J/m2), resulted in induction of p53 protein, which was suppressed in ATM gene carriers and homozygotes. The effect was depending on the proportion to the ATM gene dose. ATM-heterozygous cells vary also by greater proliferation in response to benzopyrene (BP) challenge in vitro. The battery of genes induced by polycyclic aryl hydrocarbons influences the cell proliferation and oxidation potential, that is the parameters which are changed in ATM homozygous cells. It is suggested that the impact of the BP-induced proteins on cell proliferation may be enhanced in ATM-heterozygous cells.
73 Poster Session Poster N° 37
DETECTION OF DNA DAMAGE IN B6C3F1 MALE MICE EXPOSED TO CHOSEN ANILINE DERIVATIVES Przybojewska Barbara Institute of Occupational Medicine, Lodz, Poland
The effects of many genotoxic compounds are tissue and cell-type specific. Thus it is important to utilize techniques which can directly detect DNA damage in individual cells. The single cell gel electrophoresis ('comet') assay is capable to detect DNA single-strand breaks and alkali labile sites in individual cells. The importance of this technique lies in its ability to detect intercellular differences in DNA damage in virtually any eucaryote cell population, and in its requirement for extremely small cell samples. The aim of this study was to investigate the influence of two anilin derivatives: 2,4-dimethylaniline and 2,4,6-trimethylaniline on DNA of B6C3F1 male mice using the 'comet' assay. The experiment was conducted as described by Singh et al. (1988). Results of the study are summarized in Table 1. The study indicates that 2,4-dimethylaniline and 2,4,6-trimethylaniline increase the extent of DNA migration in bone marrow cells of mice, which reflects induced DNA damage.
Table 1. Bone marrow cells from B6C3F1 mice treated with chosen aniline derivatives* ^ Concentration Mean total % cells with mg/kg(%LD5o) image lenghf (|im) migration Negative control (0.5% Tween 80) 27 ± 9 0 2,4-dimethylaniline 200 (80) 124 ± 17b 80 2,4,6-trimethylaniline 300 (80) 188 ± 72b 92 Cyclophosphamide 150 90 + 18b 76
* Results are presented as the means of two independent experiments (6 mice per dose in each; 100 cells ± S.D. per mouse) a Diameter of the nucleus plus migrated DNA. bp<0.01 (Student's test)
74 Poster Session PL9800390 Poster N° 15
ACTIVITY OF TOPOISOMERASE I AND II IN X-RAY-SENSITIVE MAMMALIAN CELL MUTANTS I. Rahden-Staron1. M.Szumilo1, H. Czeczot1, M.Z. Zdzienicka2. 1 Department of Biochemistry, Warsaw Medical School, Warsaw, Poland. 2 MGC-Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Leiden, The Netherlands.
DNA topoisomerases are nuclear enzymes capable of sensing and changing the topology of DNA. Therefore they are very attractive components of the pathways for repair of different DNA lesions. A possible involvement of DNA topoisomerases I and II in repair of X-ray induced DNA damage has been suggested. In order to prepare the groundwork for studies on the involvement of topoisomerases in repair of 7-radiation induced DNA damage, different X-ray sensitive mammalian cell mutants belonging to several complementation groups were examined for their sensitivity to inhibitors of topo I and II, camptothecin (CPT) and etoposide (VP16), respectively. In general, the mutants defective in single-strand break repair showed an increased sensitivity to CPT, whereas the mutants defective in double-strand break repair, displayed hypersensitivity to VP16. Our results suggest that DNA damage (strand breaks) generated by topoisomerase II inhibitor and X- ray are repaired via a common pathway. Therefore, the involvement of DNA repair mechanisms in cell killing by topo I and topo II inhibitors, and DNA single- and double-strand breaks may be central for their activities. To delineate whether the observed hypersensitivity to CPT/VP16 is correlated with the level of the topoisomerases I and II, we examined the intracellular levels of both topoisomerases in different mutants theorizing that they might be determinants of the cellular sensitivity to ionizing radiation and oxidative damage, as well as to topoisomerases inhibitors. We found that the activity of topo II remained similar in all the tested mutants and their parental wild-type cells, independently of their differential hypersensitivity to VP16. However, a clear inverse relationship between sensitivity to CPT and the level of topo I activity was observed.
75 Poster Session Poster N° 32
CHROMOSOME INSTABILITY IN HEAD AND NECK CANCER PATIENTS Kamila Schlade1, Robert Bartusiak1, Tomasz Krecicki2, Maria Sasiadek1 'Genetic Department, Wroclaw University of Medicine, Wroclaw, Poland department of Otolaryngology, Wroclaw University of Medicine, Wroclaw, Poland
Carcinogenesis is a multistep process resulting from the serial genetic mutations, which may occur spontaneously, but most of them are induced by mutagens. Although the whole human population is exposed to mutagenic agents only a small proportion develops cancer. This supports the thesis that the individual susceptibility to carcinogens is a very important factor in the process of cancer development. It was proved that chromosomal instability after damage induction in the S-G2 phase of the cell cycle is related to cancer susceptibility. One of the most widely used assays in studying this phenomenon is bleomycin test. In this paper the complex analysis of bleomycin - induced chromosome damage in the HNSCC patients in comparison to the control subjects is presented. Taking into account the percentage of the damaged cells and break per cell values, the signifcant increase in susceptibility to bleomycin - induced chromosome damage was observed in the HNSCC patients. The results were also analysed in regard to the clinical stage of the disease. The results suggest that the chromosome instability does not correlate with the progression of the disease. Therefore it can be assumed that bleomycin - induced chromosome aberrations reflect the general instability in cancer patients' genomes, but do not constitute the by- product of tumour progression.
76 Poster Session PL9800391 Poster N° 8
ROLE OF OUTER MEMBRANE PROTEASE OmpT IN REGULATION OF EXCISION REPAIR IN Escherichia coli Milena Sedliakova, Frantisek Masek, Viera Slezarikova and Miroslav Pirsel Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia,
It has been shown that UvrABC endonuclease can attack undamaged DNA and suggested that its activity may be regulated by OmpT protease (Caron and Grossman 1988, Nucl. Acids Res., 16, 10903). This protease cleaves UvrB protein in vitro, which renders the endonuclease inactive in excision (ibid.). The OmpT belongs among heat shock proteins, some of them being induced by various stress conditions including starvation and UV irradiation. As found earlier (Sedliakova et al. 1981, Biophys. J. 36, 429), such conditions, applied prior to UV irradiation, reduce pyrimidine dimer excision (PDE). Therefore, we have now followed PDE in the three types of pre-UV-stressed E.coli, namely: ompT +; ompT; ompT' transformed with plasmid containing ompT gene. We have found that the PDE reduction is ompr-dependent. Our data suggest: i/ that in UV irradiated cells the OmpT protease may be induced (and activated?) to terminate the period of excision repair; ii/ that in pre-UV-stressed (thus preinduced) cells the PDE may be terminated prematurely.
77 Poster Session Poster N° 5
EXAMINATION OF THE ROLE OF DNA POLYMERASE PROOFREADING IN THE MUTATOR EFFECT OF MISCODING tRNAs Malgorzata Shipska, Angela G. King, Louise I. Lu, Rose H. Lin, Emily Mao, Chantal A. Lackey, Ju-Huei Chang, Claudia Baikalov and Jeffrey H. Miller Department of Microbiology and Molecular genetics and the Molecular Biology Institute, Universuty of California, Los Angeles, CA 90095
We have previously described* E. coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the mutator effect is exerted by generating a mutator polymerase. We have suggested that the proofreading subunit of polymerase III, e, is the most likely candidate. In this study, we have constructed 16 altered mutD genes by replacing each aspartic acid codon with glycine codon in the dnaQ gene encoding e. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one - a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the 7 histidine codons in the mutD gene with glycine codons and found that in 2 cases a strong mutator phenotype results. The implication of these findings for our hypothesis concerning the role of the e subunit in the mutator effect of misreading tRNAs is discussed.
* Slupska, M.M., Baikalov, C, Lloyd, R. & Miller, J.H. (1996). Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis. Proc. Nat. Acad. Sci. USA 93, 4380-5
78 Poster Session Poster N° 7
PROTECTING EFFECT OF UmuD' ON PLASMID DNA E. Speina. J. Ciesla and C. Janion Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
UmuD'C, the E. coli SOS mutagenic proteins, play a crucial role in damage inducible mutagenesis. It is widely accepted that UmuD'C in complex with RecA facilitate DNA polymerase III holoenzyme to replicate damaged DNA past the non-coding lesions, in error-prone process. However , UmuD'C (or perhaps UmuD' alone) may participate in DNA repair in error-free process and improve DNA integrity. We purified UmuD' protein from E. coli BL(ADE3)/pEC48 strain (from R. Woodgate collection). Plasmid DNA stored in TE buffer is converted slowly from circular covalently closed (ccc) to the open circular (oc) and linear (I) forms. We have found that addition of UmuD' protein to plasmid DNA have a protecting effect on its integrity and prevents the ccc—>oc—>l conversions. However, this effect is non-specific, since it occurs with the same range, when plasmid DNA is native or modified by introduction of 8-oxoguanine or apurinic sites.
79 Poster Session Poster N° 33
MICROSATELLITE INSTABILITY IN THYROID CARCINOMA Ewa Jankowska1, Tadeusz Dobosz2, Tadeusz Lukienczuk3, Maria Sasiadek1 'Genetic Department, Wroclaw University of Medicine, Wroclaw, Poland department of Forensic Medicine, Wroclaw University of Medicine, Wroclaw, Poland 3I Chair and Clinic of Surgery, Wroclaw University of Medicine, Wroclaw, Poland
Thyroid tumours form a convenient model for studying the molecular mechanisms involved in the process of multistep carcinogenesis, because there is a wide spectrum of changes observed - ranging from benign adenomas to well differentiated cancers and anaplastic carcinomas. Microsatellite instability is a recently discovered molecular landmark of mutations in DNA mismatch repair genes, leading to the so called Replication ERror (RER) tumour phenotype. The impaired effciency of DNA repair mechanisms promotes carcinogenesis. The aim of the presented study was to investigate, whether microsatellite instability (MSI) and loss of heterozygosity (LOH) are frequent phenomena in thyroid tumours. We analysed DNA isolated from the benign adenomas (10 cases) and cancers (7 cases) in comparison to paired lymphocyte DNA, as well as DNA isolated from healthy thyroid tissue (5 cases) in comparison to paired muscle DNA. Seven, randomly chosen, trinucleotide repeat markers were analysed in each case. There were neither MSI nor LOH observed in healthy thyroid tissue. In the group of benign adenomas only in 1 out of 10 cases LOH (in 2 loci) was observed. In the group of thyroid carcinomas LOH was observed in 2 out of 7 cases. No example of MSI in the whole analysed material was observed. The results confrm the recent data indicating the absence of MSI in thyroid carcinomas.
80 Poster Session Poster N° 2
THE DIFFERENCE IN THE FIDELITY OF DNA SYNTHESIS BETWEEN LEADING AND LAGGING STRANDS REPLICATION IN E. COLI STRAINS Magdalena Maliszewska Tkaczyk1. Piotr Jonczyk1, Malgorzata Podsiadla1, Roe) M. Schaaper2 and Iwona J. Fijalkowska1 'institute of Biochemistry and Biophysics, PAS 02-106 Warsaw, Poland; 2National Institute of Environmental Health Sciences, Research Triangle Park, NC USA
To improve our understanding of the role of DNA replication in mutagenesis we investigated the mutagenic potentials of leading and lagging strands DNA replication in the model organism Escherichia coli. In order to address this question, we constructed a new system that allows examination of mutagenesis of the same target gene in two orientations with the respect to the origin of DNA replication, such that a given DNA sequence would be replicated as a leading strand in one orientation and as a lagging strand in the other. For this purpose 4 pairs of strains, differing in the orientation of a mutational target, the lac operon, with respect to the origin of chromosomal DNA replication have been constructed. To measure the specificity and frequency of mutagenesis a set of lacZ alleles constructed by Cupples and Miller (1989) have been used. Our results obtained with mutator strains impaired in mismatch repair or proofreading activity indicate that there is a difference in the fidelity of replication between the two replicating DNA strands. The differences in the level of mutagenesis between the two strands were observed both, for transitions and transversions. Arguments are presented that, in contrast to previous conclusions from plasmid-based systems, on the E. coli chromosome lagging-strand replication may be more accurate than leading-strand replication.
81 PL9800392 Poster Session Poster N° 19
EFFECT OF DUAL MMS+UV TREATMENT ON DNA AND RNA SYNTHESIS IN MAMMALIAN CELLS Hana Vargova, Miroslav Pirsel Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia,
In mammalian cells, DNA damage caused by UV and methylmetanesulfonate (MMS) is repaired by NER and BER, respectively. Cyclobutane pyrimidine dimers (CPDs) are repaired faster in the active region than in the non-transcribed strand and in genome overall. Moreover,in hamster cells they are not removed from the inactive DNA. Contrary, MMS adducts are repaired efficiently and unpreferentially. The repair pattern depends on several factors: type of lesion, structural changes in DNA, thermodynamic changes, etc. We focused our interest on the comparison of the two types of repair mechanisms working simultaneously. We measured the effect of dual MMS+UV treatment on DNA and RNA synthesis in human primary fibroblasts (MUF), Chinese hamster ovary cells (CHO9) and hamster mutant cell line (ERCC3-) transfected with the human homolog - XPB gene (pCDl). DNA inhibition after MMS+UV reflects that observed after single UV irradiation in all three types of cells. It seems that cells react to quality not quantity of damage. Different situation is seen in the RNA synthesis inhibition. Concerning the fact that CPD blocks all types of RNA polymerases in mammalian cells and only RNA pol II can act thanks to preferential repair of active genes we conclude that MMS treatment has additive effect on the inhibition of RNA synthesis to UV. It might be that BER postpones the attachment of DNA to the NER factories at nuclear matrix. Since the reaction of the pCDl transfectant is very similar to that observed in MUF cells we suggest that ERCC3 gene product has probably additional role (e.g., in replication) next to its function in TFIIH complex.
82 Poster Session PL9800393 Poster N° 6
DIFFERENT RESPONSES OF E. COLIAB1157 STRAINS BEARING TN70, TN10KANAND TN9 TRANSPOSONS AFTER HALOGEN LIGHT AND UV LIGHT IRRADIATION Anna Wqjcik and Celina Janion, Department of Molecular Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
Uncovered halogen lamps are efficient sources of far and near UV radiation including UVC (190-280 nm), UVB (280-320 nm), and UVA (320-400 nm). Recently we have shown that the induction of mutations, the mutational specifity and the MFD effect observed after halogen light irradiation in Escherichia coli K-12 AB1157 strain have the same characteristic features as those seen in Escherichia coli B strains after irradiation'with 254 nm UV light, [Mutation Research 390 (1997) 85- 92]. The Tn/0 and TnlOkan transposons are commonly used as a tool for construction of various bacterial mutants. However, we have found that introduction of the Tn/0 and TnlOkan transposons to AB1157 strains makes bacteria more sensitive to, and less mutable by halogen light and UV light irradiation. Both these phenomena are observed regardless of the transposon location on the chromosom. We have tested five E. coli AB1157 strains bearing TnlOtet transposons placed in: 2 min (leuv.TnlO), 25 min (zc///7::Tn/0), 58 min (srlC::Tnl0), 64 min (near endA) and 92 min (malE::Tnl0); and one TnlOkan transposon placed in 14.5 min {zbd3104::Tnl0kan) on the linkage map. Increased sensitivity and decreased mutability after halogen light irradiation seem to be independent on tetracycline resistance proteins production since they appear regardless of the presence of antibiotic in media and these effects are the same when tetracycline resistance was replaced by kanamycine resistance. Our results point that the effects caused by the Tn70 transposons are umuDC dependent since (i) they are suppressed when bacteria are transformed with plasmid pGW2123, pGW2101, pGW2122 and pGW2020 carrying umuD'C; umuDC, umuD' and umuD, respectively (ii) they are not observed in the strain in which the umuDC operon was deleted and (iii) there is no difference in the level of umuDC- independent mutations induced by ethyl methanesulfonate in AB1157 strains bearing or not the Tn/0 transposons. The phenomena which occur after halogen light irradiation in AB1157 strains carrying Tn/0 transposons are not observed in strains with the Tn9 transposon (malB::Tn9).
83 PL9800394 Poster Session Poster N° 17
CHROMATIN RECONSTITUTED WITH XENOPUS OOCYTE EXTRACTS: AN IN VITRO MODEL TO STUDY DNA DAMAGE AND REPAIR Piotr Widiak Department of Experimental and Clinical Radiobiology, Institute of Oncology, Gliwice, Poland.
In the nucleus of eucaryotic cells DNA is packaged into nucleoprotein complex termed chromatin, that provides the compaction and organization of DNA for transcription, replication and repair processes. The fundamental unit of the chromatin is nucleosome. The formation and repair of DNA damage is strongly influenced by the presence of nucleosomes. Reconfiguration of nucleosomes takes place during DNA repair to increase accessibility of damage to repair enzymes. Reconstitution of nucleosomes after DNA repair is then necessary to recover primary chromatin structure. Now it becomes clear that study of DNA repair on chromatin templates is necessary to get better understanding of this process in vivo. To reconstitute chromatin template in vitro one can use salt gradient method to assembly nucleosomes from DNA and purified histones. Alternatively it is possible to assembly regularly spaced nucleosomes using cell-free extracts enriched in histones and assembly factors. Here I utilized minichromosomes assembled with Xenopus laevis oocyte extracts as templates to study formation and repair of UV-induced DNA photoproducts. Circular plasmid DNA was irradiated with different doses of UV-light (254 nm) and DNA adducts were assayed using 32P- postlabeling method. DNA circles were incubated with the extracts and an ATP-regenerating system. The influence of DNA lesions on efficiency and kinetics of chromatin assembly was then assayed. To characterize repair of the damage in oocyte extracts I have assayed nicking of the template (incision/excision activity) and incorporation of DNA precursors into reconstituted minichromosomes (resynthesis/ligation activity). Xenopus oocyte extracts efficiently assembled nucleosomes on the lesion-containing plasmid DNA. Repair of UV-induced damage took place simultaneously with this chromatin reconstitution.
84 Poster Session Poster N° 28
OXIDANT STRESS INDUCED AMPLIFICATION OF PROTO- ONCOGENE c-Ha-ras AND TRANSFORMATION OF EPIDERMAL 3T3 CELIS H. Wierzbicka Department of Biochemistry and Biotechnology, University of Agriculture, Poznan, Poland
Oxidative damage to DNA leading to the formation of 8-oxo-dG can be important in mutagenesis and carcinogenesis. The mutagenetic potential of 8-oxo-dG is reflected in miscoding properties. To establish either the mutagenic or carcinogenic potential of 8-oxo-dG in vivo, recombinant plasmid pSV2-neo containing the human c-Ha-ras proto-oncogene was treated with hydrogen peroxide (0.05 - 0.5 M) prior to transfection in mouse NIH 3T3 cells. Molecular analysis of the ras-(8- oxo-dG) transformed NIH 3T3 cells has shown elevated levels of normal p21-Ha-Ras protein up to ten folds over the spontaneous background. These results suggest that overexpression of normal p21-Ha-Ras protein in consequence of oxidative damaged base 8-oxo-dG in DNA could contribute to oxidant tumour promotion.
H.W. acknowledges with gratitute the Fellowship provided by the Japanese Foundation for Promotion of Cancer Research, Tokyo
85 •II ••• lllll IIIU Hill ••!•• Illll Hill Mil Ilfti PL9800395 Poster Session Poster N° 27
RADIATION-INDUCED ABERRATIONS IN HUMAN CHROMOSOMES 1 AND 2 ANALYZED BY WHOLE CHROMOSOME PAINTING Andrzej Wojcik1. Christian Strefter 'institute for Chemistry and Nuclear Technology, Warsaw, Poland. institute for Medical Radiation Biology, Essen, Germany.
Since the early days of radiation cytogenetics efforts have been made to answer the question of whether specific chromosomes or chromosomal sites are more frequently involved in chromosomal aberrations than expected on the basis of a random aberration distribution. Because chromosomal rearangements are known to occur in the course of a malignant cell transformation, the detection of chromosomes or chromosomal regions particularly susceptible to damage induced by ionizing radiation may be important in gaining a more complete understanding of radiation-induced carcinogenesis. Using the technique of whole chromosome painting we have investigated the frequencies of radiation-induced aberrations occuring in chromosomes 1 and 2 of human peripheral lymphocytes. In parallel, unstable-type aberrations were analyzed in Giemsa-stained chromosomes and the yields compared to those obtained from painted chromosomes. Emphasis was laid upon comparing the results of two individual donors. Generally, more translocations were found in chromosome 1 than in chromosome 2. All other aberrations were randomly distributed. However, a strong variability between the results of individual experiments were observed. When all aberration types were pooled, no statistically significant difference between the chromosomes 1 and 2 was detectable.
86 Poster Session PL9800396 Poster N° 26
APPLICATION OF THE COMET ASSAY FOR MONITORING DNA DAMAGE IN WORKERS EXPOSED TO CHRONIC LOW DOSE IRRADIATION Maria Wojewodzka', Marcin Kruszewski1, Teresa Iwanehko1, Andrew R. Collins2. 'institute of Nuclear Chemistry&Technology, Warsaw, Poland 2Rowett Research Institute, Greenburn Road, Bucksburn, AB2 9SB, Aberdeen, Scotland
We examined a group of people professionally at risk of exposure to low doses of ionizing radiation (altogether 49 individuals). Age, smoking habits, use of therapeutic drugs, work-related exposure to hazardous agents, previous exposures to diagnostic X-rays such as patient and nuclear medical examination were registered. For each individual the occupational radiation burden received over the past period of 5 years was taken form the official personal records based on film dosimetry controlled every month. A matched group of controls was chosen among the administrative employees (40 individuals). The mean age of the studied population at the time of blood sampling was 50 years (range 24- 69). The individuals were divided into groups according to risk of exposure, smoking habits and gender. We compared the mean tail moments and % of DNA in the tail without enzyme treatment, with endonuclease III and FPG (formamido pyrimidine - DNA - glycosylase) in tested groups. There was a significant difference (by both above mentioned nonparametric tests) between the control and hazard groups without enzyme treatment but the level of the oxidative base damage was the same in both groups. Higher DNA damage was also found for men than for women. There was no relation of DNA damage to age and smoking habits notwithstanding the enzyme treatment. Additionally, analysis of distributions of tail moment values pointed to a considerable individual diversity even in the control group. Therefore, further investigations are necessary of the stability of the comet assay as biological dosimetry method; the results obtained so far warrant such investigations.
87 LIST OF PARTICIPANTS List of Participants
Adamczyk Malgorzata Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Anuszewska Elibieta Drug Institute, Chetmska 30/34, 00-725 Warsaw, Poland fax: (48-22) 41-06-52
Baer-Dubowska Wanda Department of Pharmaceutical Biochemistry K.Marcinkowski University of Medical Sciences, 60-780 Poznan, Grunwaldzka 6 Poland fax: (48-61) 856-95-66 e-mail: [email protected]
Baranowska Hanna Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawihskiego 5A, 02-106 Warsaw, Poland fax: (48) 39-12-16-23 e-mail: [email protected]
Barbin Alain Unit of Environmental Carcinogenesis, IARC Cours Albert Thomas 150 Lyon 69372,08 Rhone-Alpes France fax: (334) 72-73-83-29 e-mail: [email protected]
91 List of Participants
Bartosova Zdena Cancer Research Institute, SAS, Spitalska 21 812 32 Bratislava, Slovakia fax:(421)7 5214604, e-mail: [email protected]
BebenekAnna Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Bebenek Katarzyna National Institut of Health P.O. Box 12233 Research Triangle Park, N.C. 27709 USA fax:(919)541-7613 e-mail: [email protected]
Boiteux Serge Commissariat a L'Energie Atomique Centre de Etudes de Fonteney-aux-Roses 60-68 Avenue du General Leclerc 92265 Fonteney-aux-Roses France fax:(331)46-54-88-59 e-mail: [email protected] von Borstel Robert C. Dept. of Biological Sciences University of Alberta Edmonton, Alberta T6G 2E9 Canada fax: (403) 492-1903 e-mail: [email protected]
92 List of Participants
Bridges Bryn University of Sussex Brighton BN19RR United Kingdom fax: (44-1273) 67-81-21 e-mail: [email protected]
Brozmanova Jela Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax: +421-7-5214604, e-mail: [email protected]
Cebulska-Wasilewska Antonina Radiation and Environmental Biology Department, Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Krak6w, Poland fax:(012)37 54 41
Ciesielski Arkadiusz Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Ciesla Jaroslaw Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
93 List of Participants
CieSla Zygmunt Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Chovanec Miroslav Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax: +421-7-5214604 e-mail: [email protected]
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Drake John W. Lab. Molecular Genetics, ES-01, NIEHS P.O. Box 12233, Research Triangle Park NC 27709-2233, USA fax:(919)541-7613 e-mail: [email protected]
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Felczak Magdalena Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
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Fikus Marta U. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48)39-12-16-23 e-mail: [email protected].
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Fridrichova Ivana Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax:+421-7-5214604
Fuschs Robert P. Groupe de Cancerogenese et de Mutagenese Moleculaire et Structurale, I.B.M.C. du CNRS, 15 rue Descartes F-67084 Strasbourg France fax: (333) 88-65-53-43 e-mail: [email protected]
Gabelova Alena Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax: +421-7-5214604
Gawel Damian Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Gniazdowski Marek Zaktad Chemii Og61nej L6dzka Akademia Medyczna Lindleya 6 90-131 L6dz Poland fax: (48-42) 78-42-77
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Gra.ziewicz Maria A. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Gruber Beata Drug Institute, Chelmska Str. 30/34, 00-725 Warsaw, Poland fax: (48-22) 41-06-52
Grzesiuk Elzbieta Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawiriskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Halas Agnieszka Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected]
Hancock Ronald Centre de RechercheL'Hotel - Dieu De Quebec 11,Cote Du Palais Quebec Canada G1R. 2J6 fax:(418)691-5439 e-mail: [email protected]
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Hayatsu Hikoya Faculty of Pharmaceutical Sciences Okayama University 1-1-1 Tsushimanaka Okayama-shi, Okayama 700 Japan fax: (086-) 254-2129 e-mail: hayatsu@ph2ews 1 .okayama-u.ac.jp
Jachymczyk Witold Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawiriskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected]
Jaloszynski Pawel Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60 479 Poznan, Poland fax: (48-61) 233 235
Janion Celina Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Jarocewicz Nella Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland fax: (48-22) 11-15-32 e-mail: [email protected]
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Jiricny Josef Lab. University of Zurich, Direction August Forel Str. 7 Postfach 424 CH-8029 Zurich Switzerland fax: (+41) 01 385 6204 e-mail: [email protected]
Johnson Robert T. CRC Mammalian Cell DNA Repair Group, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom fax: (44 0223)336676 e-mail: RTJU(5> Cus.ccm.ac.uk
Jonczyk Piotr Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5 A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Kleczkowska Hanna Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland fax: (48-22) 11-15-32 e-mail: [email protected]
Kleibl Karol Cancer Research Institute Slovak Academy of Sciences Spitalska21, 81232 Bratislava Slovakia fax:(421)7-52146 06 e-mail: [email protected]
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Konopa Jerzy Dept of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Narutowicza 11, 80-951 Gdansk, Poland fax: (48-58) 47-15-16
Koprowski Piotr Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Kostka Grazyna Department of Environmental Toxicology, National Institute of Hygiene, 24 Chocimska Street, 00-791 Warsaw, Poland fax: (48-22) 49-74-85
Kraszewska Elzbieta Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Kruszewski Marcin Institute of Nuclear Chemistry and Technology, Dorodna 16, 01-310 Warsaw, Poland fax: (48-22) 11-15-32 e-mail: [email protected]
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Kunkel Thomas National Institut of Health P.O. Box 12233 Research Triangle Park, N.C. 27709 USA fax:(919)541-7613, e-mail: [email protected]
KuSmierek Jaroslaw T. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawihskiego 5A,
fax: (48) 39-12-16-23 e-mail: [email protected].
Larminat Florence CNRS - Universite P. Sabatier Laboratoire de Pharmacologie et de Toxicologie Foundamentale 205, route de Narbonne 31400 Toulouse, France fax: (33) 5 61 17 59 94 e-mail: [email protected]
Larsen Anette Institute Gustave Roussy URA 147 CNRS 94805 Villejuif France fax:(331)42115276
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Loft Steffen Panum Instituttet Farmakologisk Institut Blegdamsvej 3 2200 Kobenhavn Dennmark fax: 45 35 32 76 10 e-mail: [email protected]
Maliszewska-Tkaczyk Magdalena Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawiriskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Margison Geofrey Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust Wilmslow Road, M20 9BX, Manchester United Kingdom fax: 0161 446 3109 e-mail: [email protected]
Mieczkowski Piotr Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Mielzynska Danuta Institute of Occupational Medicine and Environmental Health Kos~cielna 13 41-200 Sosnowiec Poland fax: (48-3) 66-11-20 e-mail: [email protected]
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Mullenders Leon H.F. State University of Leiden Dept.. Radiation Genetics & Chemical Mutagenesis Wassenaarseweg 72, 2333 AL., Leiden The Netherlands fax: +31 (0)71-522 16 15 e-mail: [email protected]
Niedzwiedl Wojciech Radiation and Environmental Biology Department, Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Krakdw, Poland fax: (48-12) 37 54 41 e-mail: [email protected]
Nivard Madelaine State University of Leiden Dept.. Radiation Genetics & Chemical Mutagenesis Wassenaarseweg 72, 2333 AL., Leiden The Netherlands fax:+31 (0)71-522 16 15 e-mail: [email protected]
Nowicka Adrianna Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
O'Connor Timothy Dept. Of Biology, Beckman Research Institute City of Hope National, Medical Centre Duarte Road, Duarte, CA 1010 USA fax:(818)358-7703 e-mail: [email protected]
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Olinski Ryszard Department of Clinical Biochemistry, Medical School, Kartowicza 24, 85-094 Bydgoszcz, Poland fax: (48-52) 41 59 33 e-mail: [email protected]
Palus Jadwiga Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Sw. Teresy 8 90-950 L6dz, Poland fax: (48-42) 31-46-10
Palut Danuta Department of Environmental Toxicology, National Institute of Hygiene, 24 Chocimska Street, 00-791 Warsaw, Poland fax: (48-42) 49-74-84
Pavlov Iouri Dept of Genetics, St. Petersburg State University, Universitetskaya Emb. 7/9 199034 Sankt-Petersburg Russia fax:(812)428-77-33 e-mail: [email protected]
Pawlak Andrzej L. Institute of Human Genetics, Pol. Acad.Sci., Strzeszynska 32, 60-479 Poznan Poland fax: (48-61) 823-32-35 e-mail: [email protected]
105 List of Participants
Pietrzykowska Irena Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Pirsel Miroslav Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax:+421-7-5214604 e-mail: [email protected]
Przybojewska Barbara Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, &w. Teresy 8, 90-950 L6dz, Poland fax:(0 42)31-46-10
Rahden-Staron Iwona Department of Biochemistry, Warsaw Medical School, Banacha 1, 02-097 Warszawa, Poland
Russev George Institute of Molecular Biology Block 21, Acad. G. Bonchev 1113 Sofia Bulgaria fax: (+359-2) 72 35 07 e-mail: [email protected]
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Rzeszowska-Wolny Joanna Dept of Experimental and Clinical Radiobiology, Institute of Oncology, Wybrzeie Armii Krajowej 15, 44-100 Gliwice, Poland fax: (48-32) 31-35-12
Salles Bernard CNRS Universite P. Sabatier Laboratoire de Pharmacologie et de Toxicologie Foundamentale 205 Route de Narbonne 31077 Toulouse France fax:+33 561175933 e-mail: [email protected]
S^siadek Maria Genetic Department, Wroclaw University of Medicine, Marcinkowskiego 1, 50-368 Wroclaw, Poland fax: (48-71) 22-49-59
Schaaper Roel National Institut of Health P.O. Box 12233 Research Triangle Park, N.C. 27709 USA fax:(919)541-7613 e-mail: [email protected]
Schlade Kamila Genetic Department, Wroclaw University of Medicine, Marcinkowskiego 1, 50-368 Wroclaw, Poland fax: (48-71) 22-49-59
107 List of Participants
Shugar Dawid Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Siedlecki Janusz Cancer Centre Institute Roentgena 5 02-781 Warsaw Poland
Singer Beatrice Donner Laboratory, University of California Berkeley, CA 94720 USA fax:(510)486-64-88
Skladanowski Andrzej Technical University of Gdansk, Narutowicza 11, 80-951 Gdansk, Poland fax: (48-58) 47-15-16 e-mail: [email protected]
Slamenova Darina Cancer Research Institute, Slovak Academy of Sciences, Spitalska21 812 32 Bratislava, Slovakia fax:+421-7-5214604
Sledziewska-Gojska Ewa Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected]
108 List of Participants
Speina Elzbieta Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected]
Slupska Malgorzata University of California, Los Angeles 405 Hilgard Ave. CA 90095 Los Angeles USA fax:(310)206-7286 e-mail: [email protected] present address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23
Stetina Rudolf Institute of Experimental Medicine, Czech Academy of Sciences, Olesnice 14 517-83 Olesnice v. Orl. horah Czech Republic fax: 0042 044 39 50 79 e-mail: [email protected]
Szyfter Krzysztof Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60 479 Poznan, Poland fax: (48-61) 233 235 e-mail: [email protected]
109 List of Participants
Tudek Barbara Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Vargova Hana Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Spitalska21, 812 32 Bratislava, Slovakia. fax: +421-7-5214604, e-mail: [email protected]
Widlak Piotr Department of Experimental and Clinical Radiobiology, Institute of Oncology, Wybrzeie Armii Krajowej 15, 44-100 Gliwice, Poland rax: (48-32) 31-35-12
Wierzbicka Henry ka Department of Biochemistry and Biotechnology, University of Agriculture, Wotynska 35 60-637 Poznan, Poland fax: (48-61) 48-71-46
Wojew6dzka Maryla Institute of Nuclear Chemistry and Technology, Dorodna 16, 01-310 Warsaw, Poland fax: (48-22) 11-15-32 e-mail: [email protected] .waw.pl
110 List of Participants
Woodgate Roger Building 6, Room 1A13 NICHD, NIH 9000 Rockville Park Bethesda, MD 20892-2725 USA fax:(301)594-1135 e-mail: [email protected]
W6jcik Andrzej Institute of Nuclear Chemistry and Technology, Dorodna 16, 01-310 Warsaw, Poland fax: (48-22) 11-15-32 e-mail: [email protected] .waw.pl
Wdjcik Anna Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawiiiskiego 5A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
Zdzienicka Malgorzata State University of Leiden Dept.. Radiation Genetics & Chemical Mutagenesis Wassenaarseweg 72, 2333 AL., Leiden The Netherlands fax:3171-522 16 15 e-mail: [email protected]
Zuk Jerzy Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5 A, Poland fax: (48) 39-12-16-23 e-mail: [email protected].
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Zapraszamy do wspotpracy z firmg PERKIN ELMER AG o/Warszawa 01-698 Warszawa ul. Smoleriskiego 4/14 tel. (22)-33.09.36 fax (22)-33.09.96 ADDITIONAL ABSTRACT
IMPLICATION OF PROLINE-2 AND LYSINE-57 IN THE VARIOUS CATALYTIC ACTIVITIES OF THE ESCHERICHA COLIFPG PROTEIN Olga SIDORKINA and Jacques LAVAL Groupe Reparation de l'ADN",URA 147 CNRS, Institut Gustave.Roussy,94805 Villejuif Cedex, France.
The E.coli Fpg protein is involved in the repair of oxidized purines generated by reactive oxygen species. It is coded for by the fpg gene. This protein has four cysteines zinc finger motive and possesses three enzymatic activities: it is i) a DNA glycosylase excising C8-oxoguanine and formamidopyrimidine residues, ii) a P-lyase nicking DNA at abasic sites by a 0-5 elmination mechanism and iii) a dRPase. The active center of the protein is located in the first 73 amino-acids of the amino terminus. The enzymatic mechanism of action of this protein implies the formation of a Shiff base intermediate between an amino group of the protein and the aldehydic group of the open form of the deoxyribose residue. We have investigated the role of Pro-2 and Lys-57 upon the various enzymatic activities of the Fpg protein by targeted mutagenesis: - The mutant Fpg proteins K57G and K57R have a comparable P-lyase activity and generate efficiently the Shiff s base complex as compared to the wild type protein, whereas the excision of 8-oxoguanine is notably decreased. The mutants Fpg K57R and K57G remove 8-oxoG residues 5 and 55 fold less efficiently than the wild type protein, respectively. - The mutant Fpg proteins P2G and P2T have no detectable AP-lyase activity but retain 15-20% of DNA glycosylase activities elminating 8-oxoG and Fapy residues as compared to the wild type protein. Hence Pro2 is mandatory for AP-lyase activity, a new and unepected result in the mechanism of action of this protein. The antimutator activity of the various mutant proteins was evaluated. These results show that it is possible to separate, in the Fpg protein, the DNA glycosylase activity from AP-nicking activity.
This work was supported by grants from CNRS, ARC, Foundation pour la Recherche Medicale and European Communities. CHANGES IN PROGRAM Wednesday 8 October 1997 Chairperson: S. Boiteux
1405 - 1435 S. Loft, Dept. of Pharmacalogy, University of Copenhagen, Denmark „ Estimation of oxidative DNA damage in man from urinary excretion of repair products"
1440. 1510 i. FeJzenszwalb, Universidade do Estado do Rio de Janeiro, Instituto de Biologia, Rio de Janeiro, Brazil „Hydrogen peroxide effects in Escherichia coti cells"
1515 - 1545 B. Bridges, University of Sussex, Brighton, Great Britain „ Oxidative DNA damage as a cause of spontaneous mutation in non-dividing cells "
1550-1605 coffee break
Chairperson: J. Drake
1605 - 1635 J. Laval, URA 147 CNRS, Inst. G. Roussy, France ,,lmplication of proline-2 and lysine-57 in the various catalytic activities of the Escherichia coli Fpg protein "
]fi40 - 1710 A. Barbin, Unit of Enviromental Carcinogenesis, IARC, Lyon, France „ Formation of DNA etheno adducts in rodents and humans and their role in carcinogenesis"
1715 - 1745 R C. von Borstel, University of Alberta, Edmonton, Canada „ Repair of radiation damage by DNA and deoxyribonucleosides"
1750 - 1820 D. Shugar, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland „ Tautomerism: is it still important? "
18 '° - departure to the welcome party
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