Characterization of RFA1-MN cells
figure posted on 2025-04-01, 19:51 authored by Ana Amiama-Roig, Marta Barrientos-Moreno, Esther Cruz-Zambrano, Luz M. López-Ruiz, Román González-Prieto, Gabriel Ríos-Orelogio, Félix Prado (A) ChEC analysis of exponentially growing cells expressing Rfa1-MN incubated in the absence or presence of 0.00...
| Autores: | , , , , , , |
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| Tipo de recurso: | conjunto de datos |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/391858 |
| Acceso en línea: | http://hdl.handle.net/10261/391858 https://digital.csic.es/handle/10261/391851 |
| Access Level: | acceso abierto |
| Palabra clave: | sister chromatid cohesion core homologous recombination active origins facilitates |
| Sumario: | figure posted on 2025-04-01, 19:51 authored by Ana Amiama-Roig, Marta Barrientos-Moreno, Esther Cruz-Zambrano, Luz M. López-Ruiz, Román González-Prieto, Gabriel Ríos-Orelogio, Félix Prado (A) ChEC analysis of exponentially growing cells expressing Rfa1-MN incubated in the absence or presence of 0.005% MMS for 2 h. Total DNA from cells permeabilized and treated with 2 mM CaCl2 for different times is shown (left). Addition of Ca2+ is required for detection of Rfa1-MN-digested DNA, as determined by running total DNA of wild-type and RFA1-MN cells growing in the absence or presence of 0.005% MMS for 2 h (right). (B) Ionizing radiation and zeocin sensitivity of RFA1-MN cells, as determined by spotting 10-fold serial dilutions of the same number of mid-log growing cells. Wild-type and rad52∆ cells were included as control. (C) Budding index and doubling time of wild-type and RFA1-MN cells. The mean and standard deviation of three (budding index) and two (doubling time) independent experiments are shown. (D) Effect of the RFA1-MN chimera in the viability of wild-type and rad52∆ cells transformed with the URA3-based plasmid pMDL5 expressing Rad52 from the GAL1 promoter in the indicated media, as determined by spotting 10-fold serial dilutions of the same number of mid-log growing cells. The lethality of the RFA1-MN rad52∆ strain was rescued with the URA3-based plasmid pDML5, which expresses RAD52 from the galactose-inducible GAL1 promoter. This strain is able to grow, even though slowly, under glucose-repressing conditions; however, this is due to basal expression from the GAL1 promoter, as indicated by the lack of growth in the presence of fluoroorotic acid (FOA) where only Ura- cells are able to grow. (E) Effect of HU and calcium in the viability of RFA1-MN cells. The rad52∆ strain was included to show the requirement of HR for the repair of HU-induced DNA lesions. The analyses were repeated at least twice with similar results. |
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