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iDamage: a method to integrate modified DNA into the yeast genome

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of...

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Autores principales: Masłowska, Katarzyna H, Laureti, Luisa, Pagès, Vincent
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846816/
https://www.ncbi.nlm.nih.gov/pubmed/31418026
http://dx.doi.org/10.1093/nar/gkz723
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author Masłowska, Katarzyna H
Laureti, Luisa
Pagès, Vincent
author_facet Masłowska, Katarzyna H
Laureti, Luisa
Pagès, Vincent
author_sort Masłowska, Katarzyna H
collection PubMed
description In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA.
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spelling pubmed-68468162019-11-18 iDamage: a method to integrate modified DNA into the yeast genome Masłowska, Katarzyna H Laureti, Luisa Pagès, Vincent Nucleic Acids Res Methods Online In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA. Oxford University Press 2019-11-18 2019-08-16 /pmc/articles/PMC6846816/ /pubmed/31418026 http://dx.doi.org/10.1093/nar/gkz723 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Masłowska, Katarzyna H
Laureti, Luisa
Pagès, Vincent
iDamage: a method to integrate modified DNA into the yeast genome
title iDamage: a method to integrate modified DNA into the yeast genome
title_full iDamage: a method to integrate modified DNA into the yeast genome
title_fullStr iDamage: a method to integrate modified DNA into the yeast genome
title_full_unstemmed iDamage: a method to integrate modified DNA into the yeast genome
title_short iDamage: a method to integrate modified DNA into the yeast genome
title_sort idamage: a method to integrate modified dna into the yeast genome
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846816/
https://www.ncbi.nlm.nih.gov/pubmed/31418026
http://dx.doi.org/10.1093/nar/gkz723
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