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1.
Nucleic Acids Res ; 49(22): 13165-13178, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34871433

RESUMO

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.


Assuntos
Domínio Catalítico , Reparo do DNA , DNA/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Domínios Proteicos , Sequência de Aminoácidos , Biocatálise , DNA/genética , DNA/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Pirimidinas/metabolismo , Homologia de Sequência de Aminoácidos
2.
Nucleic Acids Res ; 48(6): 3014-3028, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980815

RESUMO

The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.


Assuntos
Replicação do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , N-Glicosil Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Ficusina/farmacologia , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Xenopus/genética
3.
Nucleic Acids Res ; 47(6): 2922-2931, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30649547

RESUMO

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be 'handed' from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3' diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Nucleossomos/enzimologia , Cromatina/enzimologia , Cromatina/genética , DNA/química , Dano ao DNA/genética , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Esterases/genética , Humanos , Liases/química , Liases/genética , Nucleossomos/genética , Oxirredução
4.
Nucleic Acids Res ; 47(6): 3058-3071, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30698731

RESUMO

Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Dano ao DNA/genética , DNA Glicosilases/genética , Adenina/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Neoplasias Colorretais/patologia , Escherichia coli/genética , Instabilidade Genômica/genética , Guanina/análogos & derivados , Guanina/química , Humanos , Camundongos , Mutação , Estresse Oxidativo/genética
5.
BMC Med Ethics ; 22(1): 12, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563268

RESUMO

BACKGROUND: Dynamic consent has been proposed as a process through which participants and patients can gain more control over how their data and samples, donated for biomedical research, are used, resulting in greater trust in researchers. It is also a way to respond to evolving data protection frameworks and new legislation. Others argue that the broad consent currently used in biobank research is ethically robust. Little empirical research with cohort study participants has been published. This research investigated the participants' opinions of adding a dynamic consent interface to their existing study. METHODS: Adult participants in the Extended Cohort for E-health, Environment and DNA (EXCEED) longitudinal cohort study who are members of the EXCEED Public and Participant Engagement Group were recruited. Four focus groups were conducted and analysed for thematic content. Discussion topics were derived from a review of the current literature on dynamic consent. RESULTS: Participants were in favour of many aspects of a dynamic consent interface, such as being able to update their information, add additional data to their records and choose withdrawal options. They were supportive provided it was simple to use and not intrusive. Participants expressed a markedly high level of trust in the study and its investigators and were unanimously happy with their current participation. No strong support was found for adding a dynamic consent interface to EXCEED. CONCLUSIONS: Trust in the study researchers was the strongest theme found. Openness and good data security were needed to retain their trust. While happy to discuss dynamic consent, participants were satisfied with the current study arrangements. There were indications that changing the study might unnecessarily disturb their trust. This raised the question of whether there are contexts where dynamic consent is more appropriate than others. This study was limited by the small number of participants who were committed to the study and biased towards it. More research is needed to fully understand the potential impact of adding a dynamic consent interface to an existing cohort study.


Assuntos
Pesquisa Biomédica , Consentimento Livre e Esclarecido , Adulto , Estudos de Coortes , Humanos , Estudos Longitudinais , Pesquisa Qualitativa
6.
Genet Med ; 21(7): 1486-1496, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30504929

RESUMO

PURPOSE: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. METHODS: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. RESULTS: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. CONCLUSION: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Técnicas Genéticas , Células 3T3 , Animais , Teorema de Bayes , Calibragem , Simulação por Computador , Humanos , Técnicas In Vitro , Camundongos , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Nucleic Acids Res ; 45(5): 2897-2909, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-27994037

RESUMO

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.


Assuntos
DNA Glicosilases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , DNA/química , DNA/metabolismo , DNA Glicosilases/química , Humanos , Microscopia de Força Atômica , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
PLoS Genet ; 12(8): e1006208, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27513445

RESUMO

The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/genética , Proteína BRCA2/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Doxorrubicina/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Humanos , Células MCF-7 , Mitomicina/administração & dosagem , Mutação , Rad51 Recombinase/biossíntese , Radiação Ionizante , Troca de Cromátide Irmã/genética
9.
J Biol Chem ; 292(13): 5227-5238, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28184006

RESUMO

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin.


Assuntos
DNA Ligases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Nucleossomos/genética , Sítios de Ligação , Cromatina , DNA Ligase Dependente de ATP , DNA Ligases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histonas/metabolismo , Humanos , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
10.
Nucleic Acids Res ; 43(8): 4039-54, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25813041

RESUMO

G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomeric quadruplexes in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases can remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplexes. Interestingly, a hydantoin lesion at the site most prone to oxidation in quadruplex DNA is not efficiently removed by NEIL1 or NEIL3. However, NEIL1, NEIL2 and NEIL3 remove hydantoins from telomeric quadruplexes formed by five TTAGGG repeats much more rapidly than the commonly studied four-repeat quadruplex structures. We also show that APE1 cleaves furan in selected positions in Na(+)-coordinated telomeric quadruplexes. In promoter G-quadruplex DNA, the NEIL glycosylases primarily remove Gh from Na(+)-coordinated antiparallel quadruplexes but not K(+)-coordinated parallel quadruplexes containing VEGF or c-MYC promoter sequences. Thus, the NEIL DNA glycosylases may be involved in both telomere maintenance and in gene regulation.


Assuntos
DNA Glicosilases/metabolismo , Quadruplex G , Guanina/metabolismo , N-Glicosil Hidrolases/metabolismo , Regiões Promotoras Genéticas , Telômero/metabolismo , DNA/química , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Furanos/metabolismo , Guanidinas/metabolismo , Guanina/análogos & derivados , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Hidantoínas/metabolismo , Oxirredução , Potássio/química , Sódio/química , Compostos de Espiro/metabolismo , Telômero/química
11.
Proc Natl Acad Sci U S A ; 111(20): E2091-9, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24799677

RESUMO

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.


Assuntos
Dano ao DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxigênio/química , Alanina/genética , Domínio Catalítico , Análise Mutacional de DNA , Reparo do DNA , DNA Bacteriano/química , Difusão , Escherichia coli/metabolismo , Conformação Molecular , Mutação , Ligação Proteica , Estresse Mecânico
12.
Med Law Int ; 17(3): 158-182, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28943725

RESUMO

Genome-wide sequencing technologies are beginning to be used in projects that have both clinical diagnostic and research components. The clinical application of this technology, which generates a huge amount of information of varying diagnostic certainty, involves addressing a number of challenges to establish appropriate standards. In this article, we explore the way that UK law may respond to three of these key challenges and could establish new legal duties in relation to feedback of findings that are unrelated to the presenting condition (secondary, additional or incidental findings); duties towards genetic relatives as well as the patient and duties on the part of researchers and professionals who do not have direct contact with patients. When considering these issues, the courts will take account of European and international comparisons, developing guidance and relevant ethical, social and policy factors. The UK courts will also be strongly influenced by precedent set in case law.

13.
Bioinformatics ; 31(20): 3241-8, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26112289

RESUMO

MOTIVATION: The data that put the 'evidence' into 'evidence-based medicine' are central to developments in public health, primary and hospital care. A fundamental challenge is to site such data in repositories that can easily be accessed under appropriate technical and governance controls which are effectively audited and are viewed as trustworthy by diverse stakeholders. This demands socio-technical solutions that may easily become enmeshed in protracted debate and controversy as they encounter the norms, values, expectations and concerns of diverse stakeholders. In this context, the development of what are called 'Data Safe Havens' has been crucial. Unfortunately, the origins and evolution of the term have led to a range of different definitions being assumed by different groups. There is, however, an intuitively meaningful interpretation that is often assumed by those who have not previously encountered the term: a repository in which useful but potentially sensitive data may be kept securely under governance and informatics systems that are fit-for-purpose and appropriately tailored to the nature of the data being maintained, and may be accessed and utilized by legitimate users undertaking work and research contributing to biomedicine, health and/or to ongoing development of healthcare systems. RESULTS: This review explores a fundamental question: 'what are the specific criteria that ought reasonably to be met by a data repository if it is to be seen as consistent with this interpretation and viewed as worthy of being accorded the status of 'Data Safe Haven' by key stakeholders'? We propose 12 such criteria. CONTACT: paul.burton@bristol.ac.uk.


Assuntos
Acesso à Informação , Pesquisa Biomédica , Confidencialidade , Atenção à Saúde , Humanos , Pesquisa
14.
Nature ; 464(7291): 993-8, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20393554

RESUMO

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.


Assuntos
Genética Médica/organização & administração , Genoma Humano/genética , Genômica/organização & administração , Cooperação Internacional , Neoplasias/genética , Metilação de DNA , Análise Mutacional de DNA/tendências , Bases de Dados Genéticas , Genes Neoplásicos/genética , Genética Médica/tendências , Genômica/tendências , Humanos , Propriedade Intelectual , Mutação , Neoplasias/classificação , Neoplasias/patologia , Neoplasias/terapia
15.
Bioethics ; 30(3): 210-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25960157

RESUMO

Re-consent in research, the asking for a new consent if there is a change in protocol or to confirm the expectations of participants in case of change, is an under-explored issue. There is little clarity as to what changes should trigger re-consent and what impact a re-consent exercise has on participants and the research project. This article examines applicable policy statements and literature for the prevailing arguments for and against re-consent in relation to longitudinal cohort studies, tissue banks and biobanks. Examples of re-consent exercises are presented, triggers and non-triggers for re-consent discussed and the conflicting attitudes of commentators, participants and researchers highlighted. We acknowledge current practice and argue for a greater emphasis on 'responsive autonomy,' that goes beyond a one-time consent and encourages greater communication between the parties involved. A balance is needed between respecting participants' wishes on how they want their data and samples used and enabling effective research to proceed.


Assuntos
Pesquisa Biomédica/ética , Consentimento Livre e Esclarecido/ética , Autonomia Pessoal , Projetos de Pesquisa , Bancos de Tecidos , Bancos de Espécimes Biológicos , Humanos , Consentimento Livre e Esclarecido/normas , Pesquisadores
16.
Proc Natl Acad Sci U S A ; 110(35): 14314-9, 2013 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-23940330

RESUMO

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, n(th) endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/genética , Instabilidade Genômica , Transformação Celular Neoplásica/genética , Humanos , Polimorfismo de Nucleotídeo Único
17.
Radiat Phys Chem Oxf Engl 1993 ; 128: 126-133, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27818579

RESUMO

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

19.
J Biol Chem ; 289(29): 19881-93, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24891506

RESUMO

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Nucleossomos/metabolismo , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , DNA/química , DNA/metabolismo , DNA/efeitos da radiação , Dano ao DNA , DNA Glicosilases/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oxirredução
20.
BMC Public Health ; 15: 4, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25604029

RESUMO

BACKGROUND: Although smoking prevalence in England has declined, one in five adults smoke. Smokers are at increased risk of a number of diseases, including COPD which affects an estimated 1.5 million people in England alone. This study aimed to explore issues relating to smoking behaviour and intention to quit that might be used to inform the development of cessation interventions. Issues explored included knowledge of smoking related disease, with a particular emphasis on Chronic Obstructive Pulmonary Disease (COPD). Understanding around risk of disease, including genetic risk was explored, as were features of appropriate and accessible cessation materials and support. METHODS: Semi-structured interviews and focus groups were conducted with a total of 30 individuals of which 17 were smoking cessation clients and 13 were professionals working within health care settings relevant to supporting smokers to quit. A largely purposive approach was taken to sampling, and data were analysed using the constant comparative method. RESULTS: Knowledge of the smoking related disease COPD was limited. Smokers' concerns around risk of disease were influenced by their social context and were more focussed on how their smoking might impact on the health of their family and friends, rather than how it might impact on them as individuals. Participants felt the provision of genetic risk information may have a limited impact on motivation to quit. Genetic risk was considered to be a difficult concept to understand, particularly as increased risk does not mean an individual will definitely develop disease. In terms of cessation approaches, the use of visual media was consistently supported, as was the use of materials that linked directly with life experiences. Images of children inhaling second hand smoke for example, had a particular impact. CONCLUSIONS: Public health messages around the risks of smoking and approaches to quitting should continue to have an emphasis on the dangers that an individual's smoking has on the lives of the people around them. More work also needs to be done to raise awareness around both the risk of COPD in smokers and the impact this disease has on quality of life and life expectancy.


Assuntos
Motivação , Abandono do Hábito de Fumar/psicologia , Prevenção do Hábito de Fumar , Fumar/psicologia , Adulto , Inglaterra , Feminino , Grupos Focais , Predisposição Genética para Doença , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Intenção , Masculino , Doença Pulmonar Obstrutiva Crônica/etiologia , Pesquisa Qualitativa , Medição de Risco , Abandono do Hábito de Fumar/métodos
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