Einfluss von Uracil in der DNA auf die Genexpression : die Rolle der Basen-Exzisions-Reparatur

Uracil ist eine der am häufigsten vorkommenden DNA-Basenmodifikationen, die über den Mechanismus der Basen-Exzisions-Reparatur (BER) aus dem Genom entfernt wird. Im Verlauf der Reparatur dieser Läsion durch monofunktionelle Uracil-DNA-Glykosylasen (UNG1/2, SMUG1, TDG und MBD4) entstehen AP-Läsionen und Einzelstrangbrüche. Da von beiden bekannt ist, eine Blockade der Transkription verursachen zu können, wurde in dieser Arbeit der Einfluss von Uracil und dessen Exzision auf die Expression eines Gens untersucht. Dafür wurde eine effiziente Methode entwickelt, die DNA-Basenmodifikation spezifisch in den transkribierten oder nicht-transkribierten DNA-Strang eines Reporter-Vektors einzufügen. rnIn Host cell reactivation Assays konnte gezeigt werden, dass Uracil unabhängig davon, ob es mit Adenin gepaart (U:A) oder mit Guanin (U:G) eine Fehlpaarung bildet, keine direkte Blockade der Transkriptions-Maschinerie in menschlichen Zellen auszulösen vermag. Dies kann daraus geschlossen werden, dass die Expression des Reportergens der Uracil-enthaltenen Vektoren im Vergleich zu unmodifizierten Referenz-Vektoren kurze Zeit nach der Transfektion unverändert ist. Die erst mit zunehmender Inkubationszeit in den Wirtszellen progressiv abnehmende Transkription ließ vermuten, dass die intrazelluläre Prozessierung der Läsion über die BER für die verringerte Genexpression verantwortlich ist. In der Tat bewirkte der Knockdown der BER-initiierenden UNG1/2, die Uracil aus der DNA herausschneidet und damit eine AP-Läsion generiert, eine Verringerung des negativen Effektes eines U:A-Basenpaares auf die Genexpression. Dass der Knockdown der SMUG1- oder TDG-Glykosylase hingegen keine Auswirkungen zeigte, beweist, dass UNG1/2 die Hauptglykosylase für die Exzision dieser Läsion und der Auslöser der inhibierten Transkription in HeLa-Zellen darstellt. Der Zusammenhang zwischen dem Maß des Ausschnitts einer DNA-Basenmodifikation im Verlauf der BER und einer verringerten Expression des Reportergens konnte zudem am Beispiel von 5-Hydroxymethyluracil und der für diese Läsion spezifischen SMUG1-Glykosylase nachgewiesen werden. Im Falle einer U:G-Fehlpaarung besaß weder UNG1/2 noch SMUG1 oder TDG einen Einfluss auf die Rate oder das Ausmaß der mit der Zeit abnehmenden Genexpression, was die Beteiligung einer anderen Glykosylase oder eines anderen Reparatur-Mechanismus vermuten lässt. rnDie Tatsache, dass die Stärke der Gen-Suppression unabhängig davon war, ob Uracil im transkribierten oder nicht-transkribierten DNA-Strang positioniert wurde, lässt die Mutmaßung zu, dass keine Blockade der elongierenden RNA-Polymerase, sondern vielmehr ein indirekter Mechanismus der Auslöser für die verringerte Transkription ist. Dieser Mechanismus muss unabhängig von der gut untersuchten transkriptionsgekoppelten Nukleotid-Exzisions-Reparatur erfolgen, da der Knockdown des hierfür benötigten CSB-Gens keine Auswirkungen auf die Inhibition der Genexpression der Uracil-enthaltenen Vektoren hatte. Insgesamt liefert diese Arbeit neue Erkenntnisse über den Beitrag der einzelnen Uracil-DNA-Glykosylasen zur Reparatur der DNA-Basenmodifikation Uracil in humanen Zellen und zeigt, dass die BER über einen indirekten Mechanismus die Hemmung der Genexpression verursacht.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Thymine hydroperoxide as a potential source of singlet molecular oxygen in DNA

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O(2) ((1)Delta(g))] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O(2) ((1)Delta(g)). Spectroscopic evidence for generation of O(2) ((1)Delta(g)) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D(2)O enhancement of O(2) ((1)Delta(g)) production and the quenching effect of NaN(3). Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O(2) ((1)Delta(g)) derived from the H(2)O(2)/HOCl system and from the thermolysis of the N,N`-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O(2) ((1)Delta(g)) generation was obtained by chemical trapping of O(2) ((1)Delta(g)) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce(4+) ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O(2) ((1)Delta(g)) by the Russell mechanism and point to the involvement of O(2) ((1)Delta(g)) in thymidine hydroperoxide cytotoxicity. (C) 2009 Elsevier Inc. All rights reserved.

Generierung und Prozessierung oxidativer DNA-Schäden

Generierung und Prozessierung oxidativer DNA Schäden --- Ziel dieser Arbeit war es, adaptive Antworten der Zellen auf einen DNA Schädigung zu untersuchen. Hierzu wurden Experimente zur Reparatur oxidierter Basen (Substrate der Basen Exzisions Reparatur (BER)) oder von Pyrimidindimeren (Substrate der Nukleotid Exzisions Reparatur (NER)) nach einer Vorbehandlung mit DNA-schädigender Agenzien durchgeführt. Die Ergebnisse zeigten, dass sowohl eine Vorbehandlung mit einer alkylierenden als auch mit einer oxidierenden Substanz zu einer adaptiven Erhöhung des zellulären Glutathionspiegels führte, die 16 h nach der Schädigung ihr Maximum erreichte. Jedoch waren die 8-oxoG Glykosylaseaktivitäten über einen Zeitraum von 18 h konstant. Diese Effekte waren unabhängig davon, ob Maus Embryofibroblasten, primäre oder p53 profiziente menschliche Zellen verwendet wurden. Die BER war ebenfalls in keiner der verschiedenen Zelllinien signifikant verbessert. Die adaptive Antwort bezüglich der Glutathionspiegel war also nicht mit einer entsprechenden Veränderung bei der DNA-Reparatur verbunden. Folglich ist die Reparatur von oxidativen DNA-Schäden durch eine vorausgehende Schädigung nicht induzierbar. Der zweite Teil der Untersuchungen zu der Reparatur beschäftigte sich mit der NER. Hierzu wurde die Reaktivierung eines mit UVB-Strahlung geschädigten Plasmids untersucht. Als Wirtszellen fungierten primäre menschliche Fibroblasten und Keratinozyten, die entweder mit UVB vorbehandelt oder ungeschädigt waren. Auch für die NER konnte keine signifikante Beschleunigung der Reparatur von Pyrimidindimeren durch eine Vorbehandlung festgestellt werden. Die Reaktivierung erfolgte ferner unabhängig vom p53-Status der Zellen, wie Versuche mit p53-siRNA zeigten. Neben der Prozessierung war die Generierung oxidativer DNA Schäden Gegenstand der Arbeit. Die verwendete Substanz Tirapazamin (TPZ) ist ein für hypoxische Zellen selektives, neues Zytostatikum und befindet sich momentan in Phase 2/3 der klinischen Prüfung. Ziel war es die von TPZ verursachten DNA Modifikationen zu charakterisieren, sowie die Toxizität und Genotoxizität zu untersuchen. Da es Hinweise auf eine Aktivierung von TPZ über eine Oxidoreduktase (OR) gab, wurden die Experimente in Wildtyp und hOR überexprimierenden Zellen durchgeführt. Die Quantifizierung der verursachten DNA-Modifikationen zeigte, dass der von TPZ verursachte Schaden in Zellen mit hOR erhöht war. Das erhaltene Schadensprofil der durch TPZ verursachten DNA-Modifikationen war dem Schadensprofil von durch Gamma-Strahlung intrazellulär verursachten Hydroxylradikalen sehr ähnlich. Da es nach der Aktivierung von TPZ durch eine OR zu einer Abspaltung von Hydroxylradikalen kommt, bestätigte dies den vermuteten Mechanismus. Weitere Untersuchungen mit t-Butanol, einem Hydroxylradikal Fänger, ergaben eine verminderte DNA-Schädigung, was ebenfalls für eine DNA-Schädigung durch Hydroxylradikale spricht. Untersuchungen zur Mutagenität zeigten das die Mutationsrate in Zellen mit hOR um das 4 fache erhöht ist. Erstaunlich war jedoch, dass der im gleichen Ausmaß von Gamma-Strahlung verursachte DNA-Schaden für die beobachtete Toxizität dieser verantwortlich war, während bei TPZ unter den gleichen Bedingungen keine Toxizität vorlag. Erklärt werden könnte die erhöhte Toxizität und Mutagenität durch so genannte geclusterte DNA-Schäden, die von Gamma-Strahlen, nicht jedoch von TPZ gebildet werden. Nach einer verlängerten Inkubation wurde sowohl für die Toxizität als auch für die Genotoxizität erneut ein verstärkender Effekt durch die OR bestätigt. Überraschend war weiterhin die von der OR unabhängige Generierung von Doppelstrangbrüchen, für die demnach ein grundsätzlich anderer Mechanismus, wie zum Beispiel eine direkte Interaktion mit der Topoisomerase II, angenommen werden muss.

European ancestry and polymorphisms in DNA repair genes modify the risk of melanoma: A case-control study in a high UV index region in Brazil

Background: UV radiation is the major environmental factor related to development of cutaneous melanoma. Besides sun exposure and the influence of latitude, some host characteristics such as skin phototype and hair and eye color are also risk factors for melanoma. Polymorphisms in DNA repair genes could be good candidates for susceptibility genes, mainly in geographical regions exposed to high solar radiation. Objective: Evaluate the role of host characteristic.; and DNA repair polymorphism in melanoma risk in Brazil. Methods: We carried out a hospital-based case-control study in Brazil to evaluate the contribution of host factors and polymorphisms in DNA repair to melanoma risk. A total of 412 patients (202 with melanoma and 210 controls) were analyzed regarding host characteristics for melanoma risk as well as for 11 polymorphisms in DNA repair genes. Results: We found an association of host characteristics with melanoma development, such as eye and hair color, fair skin, history of pigmented lesions removed, sunburns in childhood and adolescence, and also European ancestry. Regarding DNA repair gene polymorphisms, we found protection for the XPG 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in the XPC gene (PAT+; IV-6A and 939Gln), which represent a haplotype for XPC. Melanoma risk was higher in individuals carrying the complete XPC haplotype than each individual polymorphism (OR 3.64; 95% CI 1.77-7.48). Conclusions: Our data indicate that the host factors European ancestry and XPC polymorphisms contributed to melanoma risk in a region exposed to high sun radiation. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

Effects of physical exercise training in DNA damage and repair - could the difference be in hOGG1 Ser326Cys polymorphism?

Acute physical exercise is associated with increased oxygen consumption, which could result in an increased formation of reactive oxygen species (ROS). ROS can react with several organic structures, namely DNA, causing strand breaks and a variety of modified bases in DNA. Physical exercise training seems to decrease the incidence of oxidative stress-associated diseases, and is considered as a key component of a healthy lifestyle. This is a result of exercise-induced adaptation, which has been associated with the possible increase in antioxidant activity and in oxidative damage repair enzymes, leading to an improved physiological function and enhanced resistance to oxidative stress (Radak et al. 2008). Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is involved in the base excision repair (BER) pathway and encodes an enzyme responsible for removing the most common product of oxidative damage in DNA, 8-hydroxyguanine (8-OH-G). The genetic polymorphism of hOGG1 at codon 326 results in a serine (Ser) to cysteine (Cys) amino acid substitution (Ser326Cys). It has been suggested that the carriers of at least one hOGG1Cys variant allele exhibit lower 8-OH-G excision activity than the wild-type (Wilson et al. 2011). The aim of this study was to investigate the possible influence of hOGG1 Ser326Cys polymorphism on DNA damage and repair activity in response to 16 weeks of combined physical exercise training, in thirty healthy Caucasian men. Comet assay was carried out using peripheral blood lymphocytes and enabled the evaluation of DNA damage, both strand breaks and FPG-sensitive sites, and DNA repair activity. Genotypes were determined by PCR-RFLP analysis. The subjects with Ser/Ser genotype were considered as wild-type group (n=20), Ser/Cys and Cys/Cys genotype were analyzed together as mutant group (n=10). Regarding differences between pre and post-training in the wild-type group, the results showed a significant decrease in DNA strand breaks (DNA SBs) (p=0.002) and also in FPG-sensitive sites (p=0.017). No significant differences were observed in weight (p=0.389) and in lipid peroxidation (MDA) (p=0.102). A significant increase in total antioxidant capacity (evaluated by ABTS) was observed (p=0.010). Regarding mutant group, the results showed a significant decrease in DNA SBs (p=0.008) and in weight (p=0.028). No significant differences were observed in FPG-sensitive sites (p=0.916), in ABTS (p=0.074) and in MDA (p=0.086). No significant changes in DNA repair activity were observed in both genotype groups. This preliminary study suggests the possibility of different responses in DNA damage to physical exercise training, considering the hOGG1 Ser326Cys polymorphism.

Fractional dynamics in DNA

This paper addresses the DNA code analysis in the perspective of dynamics and fractional calculus. Several mathematical tools are selected to establish a quantitative method without distorting the alphabet represented by the sequence of DNA bases. The association of Gray code, Fourier transform and fractional calculus leads to a categorical representation of species and chromosomes.

AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

SUMMARY High-risk human papillomavirus (hr-HPV) infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP). HPV frequency was 62.4% (88/141). The most common HPV were HPV16 (37%), HPV18 (16.3%) and HPV33/45(15.2%). An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%), low grade lesions (22.9%), high grade (57.1%) and carcinoma (93.1%) (p < 0.0001). A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001), hr-HPV (p = 0.01), HSIL (p < 0.0007) and malignant lesions (p < 0.0001). Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer.

DNA damage induced by acrylamide: roe of genetic polymorphisms in DNA damage levels

RESUMO:Em 1994 a acrilamida (AA) foi classificada pela IARC como um provável cancerígeno para o homem. Para além da utilização de AA em numerosas aplicações industriais, a AA está também presente numa grande variedade de alimentos ricos em amido e processados a temperaturas elevadas. Esta exposição através da ingestão de produtos alimentares despoletou elevadas preocupações ao nível do risco para a saúde pública e poderá implicar um risco adicional para o aparecimento de cancro. A glicidamida (GA), o metabolito epóxido formado a partir da oxidação da AA provavelmente através do citocromo P450 2E1, é considerada por vários estudos, o principal responsável pela carcinogenicidade da AA. Actualmente existe uma escassez de resultados relativamente aos mecanismos de genotoxicidade da AA e GA em células de mamífero. Por este motivo, o objectivo deste estudo centra-se na avaliação das consequências genéticas da exposição à AA e GA, recorrendo-se para tal ao uso de células de mamífero como modelo. Tendo como base este objectivo avaliou-se a citotoxicidade da AA e GA, através do ensaio do MTT, e realizaram-se dois testes citogenéticos, o teste das aberrações cromossómicas (CAs) e o teste da troca de cromátides irmãs (SCEs), de modo a avaliar as lesões de DNA induzidas por estes compostos em células de hamster Chinês V79. Os resultados globalmente mostraram que a GA é mais citotóxica e clastogénica do que a AA. No âmbito deste trabalho, foi também efectuada a quantificação de aductos específicos de DNA, nomeadamente N7-(2-carbamoil-2-hidroxietil)guanina (N7-GA-Gua) e N3-(2-carbamoil-2-hidroxietil)adenina (N3-GA-Ade). Os resultados obtidos permitem afirmar que os níveis de N7-GA-Gua e a concentração de GA apresentam uma relação linear dose-resposta. Foi também identificada uma óptima correlação entre os níveis de N7-GA-Gua e a frequência de troca de cromátides irmãs. Adicionalmente, e de forma a compreender os mecanismos de toxicidade da AA, estudaram-se os mecanismos dependentes da modulação do glutationo reduzido (GSH), nomeadamente da butionina sulfoximina (BSO), um inibidor da síntese de GSH, do GSH-monoetil estér (GSH-EE), um composto permeável nas células e que é intra-celularmente hidrolisado a GSH e ainda do GSH adicionado exogenamente ao meio de cultura, em células V79. Os resultados obtidos reforçaram o papel da modulação do GSH nos efeitos de citotoxicidade e clastogenicidade da AA. Para além dos estudos efetuados com células V79, procedeu-se também à determinação da frequência de SCEs, à quantificação de aductos específicos de DNA, bem como ao ensaio do cometa alcalino em amostras de dadores saudáveis expostos à AA e GA. Tanto os resultados obtidos através do ensaio das SCE, como pela quantificação de aductos específicos de DNA, ambos efectuados em linfócitos estimulados, originaram resultados comparáveis aos obtidos anteriormente para as células V79, reforçando a ideia de que a GA é bastante mais genotóxica do que a AA. Por outro lado, os resultados obtidos pelo ensaio do cometa para exposição à AA e GA mostraram que apenas esta última aumenta o nível das lesões de DNA. Outro objectivo deste trabalho, foi a identificação de possíveis associações existentes entre as lesões de DNA, quantificadas através do ensaio das SCEs e do cometa, e biomarcadores de susceptibilidade, tendo em conta os polimorfismos genéticos individuais envolvidos na destoxificação e nas vias de reparação do DNA (BER, NER, HRR e NHEJ) em linfócitos expostos à GA. Tal permitiu identificar associações entre os níveis de lesão de DNA determinados através do ensaio das SCEs, e os polimorfismos genéticos estudados, apontando para uma possível associação entre o GSTP1 (Ile105Val) e GSTA2 (Glu210Ala) e a frequência de SCEs. Por outro lado, os resultados obtidos através do ensaio do cometa sugerem uma associação entre as lesões de DNA e polimorfismos da via BER (MUTYH Gln335His e XRCC1 Gln39Arg) e da via NER (XPC Ala499val e Lys939Gln), considerando os genes isoladamente ou combinados. Estes estudos contribuem para um melhor entendimento da genotoxicidade e carcinogenicidade da AA e GA em células de mamífero, bem como da variabilidade da susceptibilidade individual na destoxificação e reparação de lesões de DNA provocadas pela exposição a estes xenobióticos alimentares. ----------- ABSTRACT:Acrylamide (AA) has been classified as a probable human carcinogen by IARC. Besides being used in numerous industrial applications, AA is also present in a variety of starchy cooked foods. This AA exposure scenario raised concerns about risk in human health and suggests that the oral consumption of AA is an additional risk factor for cancer. A considerable number of findings strongly suggest that the reactive metabolite glycidamide (GA), an epoxide generated presumably by cytochrome P450 2E1, plays a central role in AA carcinogenesis. Until now there are a scarcity of results concerning the mechanisms of genotoxicity of AA and GA in mammalian cells. In view of that, the study described in this thesis aims to unveil the genetic consequences of AA and GA exposure using mammalian cells as a model system. With this aim we evaluated the cytotoxicity of AA and GA using the MTT assay and subsequently performed two cytogenetic end-points: chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), in order to evaluate DNA damage induced by these compounds in V79 Chinese hamster cell line. The results showed that GA was more cytotoxic and clastogenic than AA. Within the scope of this thesis the quantification of specific DNA adducts were also performed, namely N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade). Interestingly, the GA concentration and the levels of N7-GA-Gua presented a linear dose-response relationship. Further, a very good correlation between the levels of N7-GA-Gua and the extent of SCEs were observed. In order to understand the mechanisms of AA-induced toxicity, the modulation of reduced glutathione (GSH)-dependent mechanisms were studied, namely the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, of GSH-monoethyl ester (GSH-EE), a cell permeable compound that is intracellularly hydrolysed to GSH and also of GSH endogenously added to culture medium,z in V79 cell line. The overall results reinforced the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA.Complementary to the studies performed in V79 cells, SCEs, specific DNA-adducts and alkaline comet assay in lymphocytes from healthy donors exposed to AA and GA were also evaluated. Both, the frequency of SCE and the quantification of specific GA DNA adducts, produced comparable results with those obtained in V79 cell line, reinforcing the idea that GA is far more genotoxic than AA. Further, the DNA damaging potential of AA and GA in whole blood leukocytes evaluated by the alkaline comet assay, showed that GA, but not AA, increases DNA damage. Additionally, this study aimed to identify associations between DNA damage and biomarkers of susceptibility, concerning individual genetic polymorphisms involved in detoxification and DNA repair pathways (BER, NER, HRR and NHEJ) on the GA-induced genotoxicity assessed by the SCE assay and by the alkaline comet assay. The extent of DNA damage determined by the levels of SCEs induced by GA seems to be modulated by GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes. Moreover, the results obtained from the comet assay suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val and Lys939Gln) genes, either alone or in combination. The overall results from this study contribute to a better understanding of the genotoxicity and carcinogenicity of AA and GA in mammalian cells, as well as the knowledge about the variability in individual susceptibility involved in detoxification and repair of DNA damage due to these dietary xenobiotics.

The role of electron transfer in DNA building blocks: evaluation of strand breaks and their implications

Dissertação para obtenção do Grau de Doutor em Engenharia Física

DNA supercoiling and its role in DNA decatenation and unknotting.

Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation.