Versatile cell-based assay for measuring DNA alkylation damage and its repair

Abstract DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA...

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Autores principales:	Yong Li, Peng Mao, Evelina Y. Basenko, Zachary Lewis, Michael J. Smerdon, Wioletta Czaja
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f1ba10e57c694615a5775bd8ddef661d
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spelling	oai:doaj.org-article:f1ba10e57c694615a5775bd8ddef661d2021-12-02T15:15:58ZVersatile cell-based assay for measuring DNA alkylation damage and its repair10.1038/s41598-021-97523-w2045-2322https://doaj.org/article/f1ba10e57c694615a5775bd8ddef661d2021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-97523-whttps://doaj.org/toc/2045-2322Abstract DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.Yong LiPeng MaoEvelina Y. BasenkoZachary LewisMichael J. SmerdonWioletta CzajaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yong Li Peng Mao Evelina Y. Basenko Zachary Lewis Michael J. Smerdon Wioletta Czaja Versatile cell-based assay for measuring DNA alkylation damage and its repair
description	Abstract DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.
format	article
author	Yong Li Peng Mao Evelina Y. Basenko Zachary Lewis Michael J. Smerdon Wioletta Czaja
author_facet	Yong Li Peng Mao Evelina Y. Basenko Zachary Lewis Michael J. Smerdon Wioletta Czaja
author_sort	Yong Li
title	Versatile cell-based assay for measuring DNA alkylation damage and its repair
title_short	Versatile cell-based assay for measuring DNA alkylation damage and its repair
title_full	Versatile cell-based assay for measuring DNA alkylation damage and its repair
title_fullStr	Versatile cell-based assay for measuring DNA alkylation damage and its repair
title_full_unstemmed	Versatile cell-based assay for measuring DNA alkylation damage and its repair
title_sort	versatile cell-based assay for measuring dna alkylation damage and its repair
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f1ba10e57c694615a5775bd8ddef661d
work_keys_str_mv	AT yongli versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair AT pengmao versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair AT evelinaybasenko versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair AT zacharylewis versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair AT michaeljsmerdon versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair AT wiolettaczaja versatilecellbasedassayformeasuringdnaalkylationdamageanditsrepair
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Versatile cell-based assay for measuring DNA alkylation damage and its repair

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