iDamage: a method to integrate modified DNA into the yeast genome
| Autor: | Luisa Laureti, Katarzyna H. Maslowska, Vincent Pagès |
|---|---|
| Přispěvatelé: | Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Ultraviolet Rays DNA polymerase DNA damage Saccharomyces cerevisiae Locus (genetics) Computational biology Genome 03 medical and health sciences chemistry.chemical_compound Plasmid Genetics Homologous Recombination 030304 developmental biology 0303 health sciences Integrases biology 030302 biochemistry & molecular biology DNA replication [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biology biology.organism_classification Cell biology Proliferating cell nuclear antigen chemistry Gene Targeting Ubiquitin-Conjugating Enzymes biology.protein Methods Online Rad51 Recombinase Genome Fungal Homologous recombination DNA DNA Damage Plasmids |
| Zdroj: | Nucleic Acids Research Nucleic Acids Research, Oxford University Press, 2019, ⟨10.1093/nar/gkz723⟩ Nucleic Acids Research, 2019, ⟨10.1093/nar/gkz723⟩ |
| ISSN: | 0305-1048 1362-4962 |
| DOI: | 10.1101/510966 |
| Popis: | Genome of all living cell can be altered in several manners: DNA lesions such as abasic sites, DNA adducts, DNA crosslinks (intra- or inter-strand), DNA-protein crosslinks, presence of ribonucleotides... These damages, if not repaired will interfere with DNA replication, altering its fidelity, or blocking it. In order to study in details the mechanisms of replication of such DNA alterations, we have developed a method by which we insert/integrate 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 yeast genome, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert two different single UV lesions into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show the involvement and the interplay of different specialized DNA polymerases in the bypass of the lesions by translesion synthesis (TLS). More interestingly, we show that the inhibition of homologous recombination, either directly (by the inactivation of rad51 recombinase) or through its control by preventing the poly-ubiquitination of PCNA (ubc13 mutant) leads to a strong increase in the use of TLS. Such regulatory aspects of the DNA damage tolerance could not have been observed with previous methods using plasmid or randomly distributed lesion and 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 allow exploration of many biological processes related to replication and repair of modified DNA. |
| Databáze: | OpenAIRE |
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