Measuring oxidative DNA damage and DNA repair using the yeast comet assay
Autor(a) principal: | |
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Data de Publicação: | 2011 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/15787 |
Resumo: | Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. |
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Measuring oxidative DNA damage and DNA repair using the yeast comet assayComet assayYeastSaccharomyces cerevisiaeOxidative stressDNA repairHydrogen peroxideSingle cell gel eletrohoresissingle cell gel electrophoresisScience & TechnologyChromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair.John Wiley and SonsUniversidade do MinhoAzevedo, F.Marques, FilipeFokt, HannaOliveira, Rui Pedro Soares deJohansson, Björn20112011-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/15787eng1097-006110.1002/yea.182020824890http://onlinelibrary.wiley.com/doi/10.1002/yea.1820/abstract?systemMessage=Wiley+Online+Library+will+be+unavailable+17+Dec+from+10-13+GMT+for+IT+maintenance.info: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:RCAAP2024-05-11T06:56:49Zoai:repositorium.sdum.uminho.pt:1822/15787Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-05-11T06:56:49Repositório 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 |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
title |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
spellingShingle |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay Azevedo, F. Comet assay Yeast Saccharomyces cerevisiae Oxidative stress DNA repair Hydrogen peroxide Single cell gel eletrohoresis single cell gel electrophoresis Science & Technology |
title_short |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
title_full |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
title_fullStr |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
title_full_unstemmed |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
title_sort |
Measuring oxidative DNA damage and DNA repair using the yeast comet assay |
author |
Azevedo, F. |
author_facet |
Azevedo, F. Marques, Filipe Fokt, Hanna Oliveira, Rui Pedro Soares de Johansson, Björn |
author_role |
author |
author2 |
Marques, Filipe Fokt, Hanna Oliveira, Rui Pedro Soares de Johansson, Björn |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Azevedo, F. Marques, Filipe Fokt, Hanna Oliveira, Rui Pedro Soares de Johansson, Björn |
dc.subject.por.fl_str_mv |
Comet assay Yeast Saccharomyces cerevisiae Oxidative stress DNA repair Hydrogen peroxide Single cell gel eletrohoresis single cell gel electrophoresis Science & Technology |
topic |
Comet assay Yeast Saccharomyces cerevisiae Oxidative stress DNA repair Hydrogen peroxide Single cell gel eletrohoresis single cell gel electrophoresis Science & Technology |
description |
Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. |
publishDate |
2011 |
dc.date.none.fl_str_mv |
2011 2011-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/15787 |
url |
http://hdl.handle.net/1822/15787 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1097-0061 10.1002/yea.1820 20824890 http://onlinelibrary.wiley.com/doi/10.1002/yea.1820/abstract?systemMessage=Wiley+Online+Library+will+be+unavailable+17+Dec+from+10-13+GMT+for+IT+maintenance. |
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 |
John Wiley and Sons |
publisher.none.fl_str_mv |
John Wiley and Sons |
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 |
institution |
RCAAP |
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 |
repository.mail.fl_str_mv |
mluisa.alvim@gmail.com |
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1817545147343175680 |