Effects of UV radiation exposure on DNA and DNA repair enzymes

Detalhes bibliográficos
Autor(a) principal: Camacho, Inês Sofia Cortes Eusébio
Data de Publicação: 2012
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/8263
Resumo: Dissertação para obtenção do Grau de Mestre em Biotecnologia
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spelling Effects of UV radiation exposure on DNA and DNA repair enzymesEndonuclease IIIPlasmid DNAProtein‐DNA complexUV radiationElectrophoretic mobility shift assaysBioelectrochemistryDissertação para obtenção do Grau de Mestre em BiotecnologiaDNA integrity in the cell is under constant threat from damaging agents of endogenous or exogenous origin, such as UV light, ionizing radiation and oxidative stress. Although the effects of these carcinogens on DNA have been extensively studied, very little is known about their effect on DNA repair enzymes. The aim of the present work was the study of the effect of UV radiation on E. coli Endonuclease III, a DNA glycosylase belonging to base excision repair system. This enzyme was homologously overexpressed and then purified with a Fe/protein ratio of 3.88 ± 0.63 (fully‐loaded form). Endonuclease III exposure to UV radiation for 45 min (19.77 kJ dose) may lead to the destruction of the Fe‐S bonds of the [4Fe‐4S] cluster or to the conversion of this center into a different redox state. Electrophoretic mobility shift assays with protein‐DNA complex showed that Endonuclease III binding to plasmid DNA promotes a retardation of the free supercoiled DNA band, indicative of Endonuclease III‐DNA complex(es) formation. These assays also showed that Endonuclease III is able to bind both linear and supercoiled plasmid DNA, although with higher affinity for the linear form. Electrophoretic mobility shift assays performed after 45 min of UV irradiation (19.77 kJ) revealed that although shift occurred, the complexes formed were unstable and dissociated during electrophoresis. Moreover, the presence of aggregates suggests the unfolding of some Endonuclease III molecules. After 6 h of UV irradiation (158.18 kJ) no complexes are formed, leading to the conclusion that Endonuclease III molecules were irreversibly damaged. The electrochemical studies were performed by cyclic and differential pulse voltammetry techniques, at room temperature and anaerobic conditions; Endonuclease III and Endonuclease IIIDNA complex were adsorbed on a bare pyrolytic graphite electrode. For the first time, the direct electrochemical response of Endonuclease III unbound to DNA was observed, with a quasi‐reversible redox couple displaying a midpoint potential of 178 ± 9 mV vs. NHE. Endonuclease III binding to plasmid DNA promotes a positive shift (19 mV vs. NHE) in the characteristic redox couple of Endo III. Protein‐DNA complex UV irradiation promotes a negative shift in its redox potential of 25 mV vs. NHE.Faculdade de Ciências e TecnologiaFolgosa, FilipeRUNCamacho, Inês Sofia Cortes Eusébio2012-12-03T10:52:19Z20122012-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/8263enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T03:40:48Zoai:run.unl.pt:10362/8263Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:18:05.755868Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Effects of UV radiation exposure on DNA and DNA repair enzymes
title Effects of UV radiation exposure on DNA and DNA repair enzymes
spellingShingle Effects of UV radiation exposure on DNA and DNA repair enzymes
Camacho, Inês Sofia Cortes Eusébio
Endonuclease III
Plasmid DNA
Protein‐DNA complex
UV radiation
Electrophoretic mobility shift assays
Bioelectrochemistry
title_short Effects of UV radiation exposure on DNA and DNA repair enzymes
title_full Effects of UV radiation exposure on DNA and DNA repair enzymes
title_fullStr Effects of UV radiation exposure on DNA and DNA repair enzymes
title_full_unstemmed Effects of UV radiation exposure on DNA and DNA repair enzymes
title_sort Effects of UV radiation exposure on DNA and DNA repair enzymes
author Camacho, Inês Sofia Cortes Eusébio
author_facet Camacho, Inês Sofia Cortes Eusébio
author_role author
dc.contributor.none.fl_str_mv Folgosa, Filipe
RUN
dc.contributor.author.fl_str_mv Camacho, Inês Sofia Cortes Eusébio
dc.subject.por.fl_str_mv Endonuclease III
Plasmid DNA
Protein‐DNA complex
UV radiation
Electrophoretic mobility shift assays
Bioelectrochemistry
topic Endonuclease III
Plasmid DNA
Protein‐DNA complex
UV radiation
Electrophoretic mobility shift assays
Bioelectrochemistry
description Dissertação para obtenção do Grau de Mestre em Biotecnologia
publishDate 2012
dc.date.none.fl_str_mv 2012-12-03T10:52:19Z
2012
2012-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10362/8263
url http://hdl.handle.net/10362/8263
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
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reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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