Study of the cellular response, signaling, and repair pathways to confront dna double-strand breaks in telophase
- Autores: Jessel Ayra Plasencia
- Directores de la Tesis: Félix Machín (dir. tes.)
- Lectura: En la Universidad de La Laguna ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Jose Antonio Pérez Pérez (presid.), Ethelvina Queralt Badía (secret.), Gerard Mazón i Busquets (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencias de la Salud por la Universidad de La Laguna
- Materias:
- Enlaces
- Tesis en acceso abierto en: RIULL
- Resumen
Cell survival depends on the genome integrity throughout the cell cycle and includes the faithful segregation of chromosomes in anaphase. DNA is continuously compromised by both exogenous and endogenous sources, such as ionizing radiation or reactive oxygen species (ROS) from the internal metabolism, respectively. In the last instance, insults to the integrity of the DNA can happen. DNA double-strand breaks (DSBs) are among the most harmful lesions that cells have to face. DNA breakage can result in severe mutations and promote genomic instability, leading to cancer, senescence, or cell death. However, cells have developed different repair pathways to confront DSBs.
DSB repair mechanisms can be classified into non-homologous end joining (NHEJ) and homologous recombination (HR). The NHEJ pathway is triggered during the G1 phase, which is characterized by the absence of a sister chromatid to act as an intact DNA template for repair and the low CDK/cyclin activity. On the other hand, HR is used from the S phase onwards, when CDK/cyclin levels rise, and chromosomes comprise two sister chromatids. NHEJ entails an error-prone mechanism since broken DNA ends are barely processed and directly re-joined, causing short insertions and deletions at the flanking sites of the DSB. On the contrary, HR is an error-free repair pathway because the broken DNA is restored by copying the nucleotide information from the homolog intact template, usually the intact sister chromatid.
However, how cells deal with DSBs at late anaphase/telophase is still unknown. These latest stages of the cell cycle are paradoxical scenarios. First, CDK/cyclin levels are still high, and this would promote HR-mediated repair. Nonetheless, sister chromatids have been previously segregated in anaphase, supporting a more important role for NHEJ since the intact template is not close and well-aligned to be invaded.
In this work, the budding yeast Saccharomyces cerevisiae has been employed as a model to determine the cellular response and the repair pathways triggered to face DSBs in telophase. Cdc15-2 conditional mutants have been used to generate stable telophase blocks where to generate single and multiple DSBs. Fluorescence microscopy analysis has uncovered: i) the approximation of the segregated DNA material, ii) the acceleration of chromosome movement, iii) the structural and dynamics changes produced in the microtubules apparatus, and iv) the generation of coalescence events between segregated sister chromatid loci. Also, cells delay the telophase-to-G1 transition in a Rad9-dependent manner, and the partial dephosphorylation of the Cin8 kinesin motor protein is relevant to promote the reversion of segregation. The molecular monitorization of a single DSB repair also showed that cells favor HR over NHEJ in a Rad9-, Mre11-, and Yku70-independent but cohesin-dependent manner.
Additionally, an experimental approximation on the HeLa cells response has also been performed. Cells delayed cytokinesis after being confronted with phleomycin-mediated DSBs at late anaphase/telophase stages. Contrary to what happened in yeast, HeLa cells did not modify the microtubular morphology. Instead, they responded to DSBs by phosphorylating the histone variant gH2A.X. Strikingly, 53BP1, RIF1, and RPA2 foci appeared simultaneously. Although previous works have described that 53BP1 and RIF1 promote NHEJ and counteract resection, telophase-damaged cells showed a pattern where both NHEJ and HR pathways seem to cooperate.
