Filogeografia do tubarão mako Isurus oxyrinchus utilizando marcadores moleculares do DNA mitocondrial

Pós-graduação em Ciências Biológicas (Genética) - IBB

Estudos da lesão ao DNA por corantes têxteis e da capacidade protetora de flavonóides empregando biossensor eletroquímico: Carolina Venturini Uliana. -

Pós-graduação em Química - IQ

Modelos mecânicos para o estudo da dinâmica do DNA

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Interaction of Organophosphorus Pesticides with DNA Nucleotides on a Boron-Doped Diamond Electrode

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Investigação da condução elétrica em fitas duplas de dna imobilizadas em eletrodos de ouro por medidas eletroquímicas

Pós-graduação em Química - IQ

Variabilidade genética e filogeografia de populações de Prochilodus lineatus (Pisces: prochilodontidae) das bacias dos rios Paraguai-Paraná e da bacia Amazônica: bases para programas de conservação e de psicultura

A espécie Prochilodus lineatus, considerada um migrador por excelência, possui intensa ocorrência durante a migração reprodutiva. São peixes de elevado valor econômico e a sua adaptação em cativeiro os tornam altamente interessantes para desenvolvimento de programas de piscicultura. O monitoramento regular das variações genéticas nos estoques é importante em programas de conservação, evitando o declínio da variabilidade genética, essencial para a conservação das espécies. No entanto, os poucos dados moleculares populacionais para Prochilodus lineatus justificam a presente proposta. Nesse sentido, tivemos como objetivo caracterizar a variabilidade genética e estabelecer as relações filogeográficas entre 6 populações de P. lineatus das bacias dos rios Paraguai (2 do rio Paraguai e 3 do rio Cuiabá, MT) e Paraná (1 do rio Mogi-Guaçu, SP), num total de 34 indivíduos. Outros 16 indivíduos dos rios: da Prata, Uruguai, Paraná, Bermejo, Paraguai, Amazonas e Madeira, cujas sequências foram obtidas do National Center for Biotechnology Information (Genbank), foram analisados, totalizando assim, 50 indivíduos. Como grupo externo foi utilizado Salminus brasiliensis. O estudo foi realizado através das análises das sequências do gene mitocondrial ATPase 8/6. O interesse do uso de um gene mitocondrial está na vasta literatura que esse possui em análises filogenéticas e na confiança que existe nos dados gerados de tais análises. O gene citado foi completamente sequenciado (842pb), as análises filogenéticas foram conduzidas pelos métodos “Neighbor Joining (NJ)”, “Minimum Evolution (ME)” e “Máxima Parcimônia (MP)”, com 1000 réplicas de bootstrap no programa MEGA 5.0. Para análises filogeográficas as sequências foram analisadas no programa TCS e no programa Arlequin. Os resultados auxiliarão em uma melhor compreensão da história evolutiva, migração... (Resumo completo, clicar acesso eletrônico abaixo)

Diversidade gênica no gênero Astyanax (Teleostei : Characidae) através da análise de PCR-RFLP de DNA mitocondrial

O gênero Astyanax é um dos mais abundantes e diversificados da família Characidae (subfamília Tetragonopterinae), com mais de 100 espécies nominais, estando amplamente distribuído na bacia do Alto rio Paraná. Esse gênero é caracterizado pela similaridade quanto à forma do corpo, além da alta variabilidade citogenética intra e interpopulações, sendo comum a ocorrência de espécies crípticas ou complexos de espécies. Devido à escassez de dados sobre genética molecular referentes a esse gênero, e a dificuldade na identificação taxonômica, fazse necessário um estudo utilizando marcadores moleculares que visem desenvolver métodos rápidos e eficientes para caracterização de espécies de Astyanax com base em análises de DNA. Em vista dessa necessidade, o presente trabalho teve como objetivo avaliar a eficiência da técnica de PCR-RFLP do gene mitocondrial Citocromo b na identificação e caracterização da variabilidade genética de cinco espécies do gênero Astyanax que ocorrem na bacia do Alto rio Paraná. O mtDNA das espécies alvo foi totalmente amplificado, num total de cerca de 1140 pares de bases (pb). As análises foram obtidas através dos haplótipos gerados com a digestão por três enzimas de restrição que clivam estes genes, sendo estas ALUI, BAMHI, e HPAII. Foram analisadas 2 populações de Astyanax paranae, A. altiparanae, A. fasciatus, A. bockmanni, e uma população de A. biotae, com amostragens variando entre 5 e 10 indivíduos de cada população. Como grupo externo foram analisadas duas populações de Astyanax ribeirae da bacia hidrográfica do rio Ribeira de Iguape. Ao final do trabalho foi possível a identificação de 4 das 6 espécies analisadas, sendo que as espécies mais divergentes são A. altiparanae e A. ribeirae, as espécies A. paranae + A. bockmanni e A. fasciatus + A. ribeirae são as mais fortemente relacionadas...(Resumo completo, clicar acesso eletrônico abaixo)

Avaliação de danos oxidativos no DNA de linfócitos e estresse oxidativo no plasma de pacientes a cirurgia sob anestesia balanceada com sevoflurano

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

The relative roles of DNA damage induced by UVA irradiation in human cells

UVA light (320–400 nm) represents approximately 95% of the total solar UV radiation that reaches the Earth’s surface. UVA light induces oxidative stress and the formation of DNA photoproducts in skin cells. These photoproducts such as pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs, and pyrimidine (6-4) pyrimidone photoproducts, 6-4PPs) are removed by nucleotide excision repair (NER). In this repair pathway, the XPA protein is recruited to the damage removal site; therefore, cells deficient in this protein are unable to repair the photoproducts. The aim of this study was to investigate the involvement of oxidative stress and the formation of DNA photoproducts in UVA-induced cell death. In fact, similar levels of oxidative stress and oxidised bases were detected in XP-A and NER-proficient cells exposed to UVA light. Interestingly, CPDs were detected in both cell lines; however, 6-4PPs were detected only in DNA repairdeficient cells. XP-A cells were also observed to be significantly more sensitive to UVA light compared to NER-proficient cells, with an increased induction of apoptosis, while necrosis was similarly observed in both cell lines. The induction of apoptosis and necrosis in XP-A cells using adenovirus-mediated transduction of specific photolyases was investigated and we confirm that both types of photoproducts are the primary lesions responsible for inducing cell death in XP-A cells and may trigger the skin-damaging effects of UVA light, particularly skin ageing and carcinogenesis.

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Bedeutung endogener Faktoren für Steady-State-Level und Reparatur oxidativer DNA-Modifikationen in Säugerzellen

Die endogene Bildung reaktiver Sauerstoffspezies (ROS) - wie beispielsweise Hydroxyl-Radikale, Superoxid-Radikalanionen, Wasserstoffperoxid und Singulett-Sauerstoff - bei essentiellen Stoffwechselreaktionen in allen aeroben Lebewesen stellt eine potentielle Gefahr für die Integrität der DNA in jeder Zelle dar. ROS generieren in der DNA unter anderem oxidative DNA-Modifikationen (zum größten Teil wahrscheinlich 8-Hydroxyguanin (8-oxoG)), welche wiederum zu einem Teil zu Mutationen führen.In dieser Arbeit wurden Untersuchungen vorgenommen, in welchem Ausmaß zum einen die Steady-State-Level oxidativer DNA-Schäden in Säugerzellen zum anderen die Reparaturgeschwindig-keiten solcher DNA-Modifikationen durch verschiedene endogene Faktoren beeinflußt werden.Im Mittelpunkt der Arbeit stand dabei die Charakterisierung der 8-Hydroxyguaninglykosylase der Säugerzellen. Sie ist das Produkt des OGG1-Gens, das erst 1997 kloniert wurde. In transfizierten Zellinien konnte durch eine konstitutive Überexpression des menschlichen OGG1-Gens demonstriert werden, daß die Reparatur von induzierten oxidativen Basenmodifikationen bis zu dreifach beschleunigt wird und daß eine Korrelation zwischen dem Grad der Überexpression und der Reparaturrate besteht. Dagegen waren die Steady-State-Level der oxidativen DNA-Schäden durch die Überexpression unbeeinflußt. Sowohl bei den spontanen Mutationsraten als auch bei den durch oxidative Schädigungen induzierten Mutationsfrequenzen konnte keine Erniedrigung bedingt durch die hOGG1-Überexpression beobachtet werden.Weitere Untersuchungen zur Bedeutung von Ogg1-Protein konnten in Mäusezellen durchgeführt werden, in denen das OGG1-homologe Mäusegen, mOGG1, homozygot inaktiviert (mOGG1(-/-)) worden war. Hierbei konnte gezeigt werden, daß in den mOGG1-defizienten Zellen im Vergleich zu den entsprechenden Wildtyp-Zellen (mOGG1(+/+)) eine Reparatur induzierter oxidativer Basenmodifikationen erst nach 8 h einsetzt, während in den Kontrollzellen schon nach 3-4 h 50 % der Modifikationen repariert waren. Die Steady-State-Level oxidativer Modifikationen in mOGG1(-/-)-Zellen waren in immortalisierten, schnell proliferierenden Mäusefibroblasten nur um den Faktor 1.4, in primären Mäusehepatocyten jedoch um den Faktor 2.5 gegenüber den Wildtyp-Zellen erhöht.Inwieweit das menschliche Reparaturprotein Xrcc1 (X-ray repair cross complementing group 1) auch an der Prozessierung oxidativer DNA-Modifikationen beteiligt ist, und ob dabei möglicherweise eine Interaktion mit Ogg1 vorliegt, wurde in der XRCC1-defizienten CHO-Zellinie EM9 untersucht. Dabei wurde ermittelt, daß weder die Steady-State-Level noch die Reparaturkinetiken der oxidativen Basenmodifikationen durch die XRCC1-Defizienz beeinflußt werden. Aufgrund weiterer Ergebnisse kann jedoch nicht ausgeschlossen werden, daß das Xrcc1-Protein zumindest am Ligationsschritt während der Reparatur oxidativer DNA-Schäden beteiligt ist.In einem weiteren Schwerpunkt der Arbeit wurde untersucht, ob Unterschiede im Steady-State-Level in Abhängigkeit von Organ-, Gewebe- und Zelltyp auftreten. Dazu wurden Untersuchungen in Bronchialkarzinom-Zellinien verschiedener Subtypen durchgeführt. Des weiteren wurde zur Frage der Zelltyp-Abhängigkeit in der menschlichen Zellinie HL60 der Einfluß des Zelldifferenzierungsstadiums auf die Steady-State-Level untersucht.

DNA block copolymers - synthesis, morphologies and applications

The last decades have witnessed significant and rapid progress in polymer chemistry and molecular biology. The invention of PCR and advances in automated solid phase synthesis of DNA have made this biological entity broadly available to all researchers across biological and chemical sciences. Thanks to the development of a variety of polymerization techniques, macromolecules can be synthesized with predetermined molecular weights and excellent structural control. In recent years these two exciting areas of research converged to generate a new type of nucleic acid hybrid material, consisting of oligodeoxynucleotides and organic polymers. By conjugating these two classes of materials, DNA block copolymers are generated exhibiting engineered material properties that cannot be realized with polymers or nucleic acids alone. Different synthetic strategies based on grafting onto routes in solution or on solid support were developed which afforded DNA block copolymers with hydrophilic, hydrophobic and thermoresponsive organic polymers in good yields. Beside the preparation of DNA block copolymers with a relative short DNA-segment, it was also demonstrated how these bioorganic polymers can be synthesized exhibiting large DNA blocks (>1000 bases) applying the polymerase chain reaction. Amphiphilic DNA block copolymers, which were synthesized fully automated in a DNA synthesizer, self-assemble into well-defined nanoparticles. Hybridization of spherical micelles with long DNA templates that encode several times the sequence of the micelle corona induced a transformation into rod-like micelles. The Watson-Crick motif aligned the hydrophobic polymer segments along the DNA double helix, which resulted in selective dimer formation. Even the length of the resulting nanostructures could be precisely adjusted by the number of nucleotides of the templates. In addition to changing the structural properties of DNA-b-PPO micelles, these materials were applied as 3D nanoscopic scaffolds for organic reactions. The DNA strands of the corona were organized by hydrophobic interactions of the organic polymer segments in such a fashion that several DNA-templated organic reactions proceeded in a sequence specific manner; either at the surface of the micelles or at the interface between the biological and the organic polymer blocks. The yields of reactions employing the micellar template were equivalent or better than existing template architectures. Aside from its physical properties and the morphologies achieved, an important requirement for a new biomaterial is its biocompatibility and interaction with living systems, i.e. human cells. The toxicity of the nanoparticles was analyzed by a cell proliferation assay. Motivated by the non-toxic nature of the amphiphilic DNA block copolymers, these nanoobjects were employed as drug delivery vehicles to target the anticancer drug to a tumor tissue. The micelles obtained from DNA block copolymers were easily functionalized with targeting units by hybridization. This facile route allowed studying the effect of the amount of targeting units on the targeting efficacy. By varying the site of functionalization, i.e. 5’ or 3’, the outcome of having the targeting unit at the periphery of the micelle or in the core of the micelle was studied. Additionally, these micelles were loaded with an anticancer drug, doxorubicin, and then applied to tumor cells. The viability of the cells was calculated in the presence and absence of targeting unit. It was demonstrated that the tumor cells bearing folate receptors showed a high mortality when the targeting unit was attached to the nanocarrier.

Einfluss von oxidativem Stress auf die DNA-Reparatur in Säugerzellen

Gegenstand dieser Arbeit war es, das Zusammenspiel zwischen DNA-Reparatur und zellulärem anitoxidativen Abwehrsystem in Melanomzellen und gesunden Hautfibroblasten näher zu untersuchen. Dabei konnte gezeigt werden, dass die dominierenden DNA-Läsionen im Falle einer Bestrahlung mit sichtbarem Licht (400 – 800 nm) Fpg-sensitive Läsionen, zu denen die Basenmodifikation 7,8-Dihydro-8-oxoguanin (8-oxoG) gehört, und im Falle der UVA-Bestrahlung Cyclobutan-Pyrimidindimere (CPDs) sind. Sowohl Melanomzellen als auch Hautfibroblasten waren problemlos in der Lage, die durch sichtbares Licht und UVA-Strahlung induzierten oxidativen DNA-Modifikationen zu reparieren. Jedoch reagierten Melanomzellen in einer adaptiven Antwort mit einer Erhöhung ihres Glutathion-Gehalts auf ein Maximum (nach circa 10 - 14 h) nach Bestrahlung mit sichtbarem Licht, wohingegen die Hautfibroblasten einen massiven Einbruch direkt nach Bestrahlung und eine extrem lange Erholungsphase über 48 h aufzuweisen hatten. Die darauffolgende Untersuchung der DNA-Reparaturkapazität der Zellen unter Bedingungen von oxidativem Stress mit vorangegangener Depletion intrazellulären Glutathions zeigten eine dramatische, nahezu vollständige Hemmung der Reparatur durch UVA- bzw. Sonnenlicht-induzierter Fpg-sensitiver DNA-Modifikationen (8-oxoG) - sowohl in Melanomzellen als auch in Hautfibroblasten. Dieser Effekt ließ sich durch den Zusatz von Dithiothreitol (DTT), nach erfolgter Bestrahlung der Glutathion-depletierten Zellen, wieder komplett revertieren. Diese Ergebnisse weisen darauf hin, dass an der Reparatur ein redoxempfindliches Protein oder zellulärer Cofaktor beteiligt sein muß. Zudem konnte durch Untersuchungen der Nukleotidexzisionsreparatur (NER) und der Einzelstrangbruchreparatur nach dem gleichen Versuchsdesign gezeigt werden, dass es sich hierbei sehr wahrscheinlich um einen für die Basenexzisionsreparatur (BER) von 7,8-dihydro-8-oxo-guanine (8-oxoG) exklusiven Effekt handelte. Zwei der wichtigsten Reparaturproteine der BER, nämlich hOGG1 und APE1, wurden anschließend auf ihre Funktionsfähigkeit hin untersucht, da es naheliegend war, dass der Reparaturhemmung ein Funktionsverlust eines dieser beiden Enzyme zugrunde liegen könnte. Im Falle des APE1-Proteins konnte dies ausgeschlossen werden, da mit Hilfe der Alkalischen Elution die volle Funktionsfähigkeit für die Reparatur von AP-Läsionen nachgewiesen werden konnte. Interessanterweise zeigte aber das hOGG1-Protein eine zwischen der dritten und vierten Stunde nach Bestrahlung Glutathion-depletierter Zellen stark abfallende Aktivität der 8-oxoG-Glykosylasefunktion. Die Western-Blot-Analyse ergab allerdings keinen Hinweis auf eine Proteinoxidation von hOGG1. Möglicherweise wird nicht hOGG1 selbst, wohl aber ein anderes, für eine konzertierte Abfolge der einzelnen Reparaturschritte entscheidend notwendiges Protein innerhalb der Zelle durch ROS leicht oxidiert. In jedem Fall bleibt festzustellen, dass Glutathion eine wichtige Aufgabe hinsichtlich einer voll funktionsfähigen Basenexzisionreparatur zuzukommen scheint. Die Ergebnisse unterstreichen die mögliche Bedeutung von oxidativem Stress für die Entstehung von Krebs durch Sonnenlicht, insbesondere durch UVA, da die durch die Strahlung (und eventuell auftretende Entzündung) gebildeten ROS nicht nur DNA-Schäden induzieren, sondern auch ihre Reparatur verhindern können.

Consequences of DNA damage for gene transcription : direct effects of modified nucleobases and the role of base excision repair = Folgen von DNA-Schäden für die Gentranskription

The presence of damaged nucleobases in DNA can negatively influence transcription of genes. One of the mechanisms by which DNA damage interferes with reading of genetic information is a direct blockage of the elongating RNA polymerase complexes – an effect well described for bulky adducts induced by several chemical substances and UV-irradiation. However, other mechanisms must exist as well because many of the endogenously occurring non-bulky DNA base modifications have transcription-inhibitory properties in cells, whilstrnnot constituting a roadblock for RNA polymerases under cell free conditions. The inhibition of transcription by non-blocking DNA damage was investigated in this work by employing the reporter gene-based assays. Comparison between various types of DNA damage (UV-induced pyrimidine photoproducts, oxidative purine modifications induced by photosensitisation, defined synthetic modified bases such as 8-oxoguanine and uracil, and sequence-specific single-strand breaks) showed that distinct mechanisms of inhibition of transcription can be engaged, and that DNA repair can influence transcription of the affectedrngenes in several different ways.rnQuantitative expression analyses of reporter genes damaged either by the exposure of cells to UV or delivered into cells by transient transfection supported the earlier evidence that transcription arrest at the damage sites is the major mechanism for the inhibition of transcription by this kind of DNA lesions and that recovery of transcription requires a functional nucleotide excision repair gene Csb (ERCC6) in mouse cells. In contrast, oxidisedrnpurines generated by photosensitisation do not cause transcriptional blockage by a direct mechanism, but rather lead to transcriptional repression of the damaged gene which is associated with altered histone acetylation in the promoter region. The whole chain of events leading to transcriptional silencing in response to DNA damage remains to be uncovered. Yet, the data presented here identify repair-induced single-strand breaks – which arise from excision of damaged bases by the DNA repair glycosylases or endonucleases – as arnputative initiatory factor in this process. Such an indirect mechanism was supported by requirement of the 8-oxoguanine DNA glycosylase (OGG1) for the inhibition of transcription by synthetic 8-oxodG incorporated into a reporter gene and by the delays observed for the inhibition of transcription caused by structurally unrelated base modifications (8-oxoguanine and uracil). It is thereby hypothesized that excision of the modified bases could be a generalrnmechanism for inhibition of transcription by DNA damage which is processed by the base excision repair (BER) pathway. Further gene expression analyses of plasmids containing single-strand breaks or abasic sites in the transcribed sequences revealed strong transcription inhibitory potentials of these lesions, in agreement with the presumption that BER intermediates are largely responsible for the observed effects. Experiments with synthetic base modifications positioned within the defined DNA sequences showed thatrninhibition of transcription did not require the localisation of the lesion in the transcribed DNA strand; therefore the damage sensing mechanism has to be different from the direct encounters of transcribing RNA polymerase complexes with DNA damage.rnAltogether, this work provides new evidence that processing of various DNA basernmodifications by BER can perturb transcription of damaged genes by triggering a gene silencing mechanism. As gene expression can be influenced even by a single DNA damage event, this mechanism could have relevance for the endogenous DNA damage induced in cells under normal physiological conditions, with a possible link to gene silencing in general.