RESUMEN
The elimination of DNA polymerase eta (pol η) causes discontinuous DNA elongation and fork stalling in UV-irradiated cells. Such alterations in DNA replication are followed by S-phase arrest, DNA double-strand break (DSB) accumulation, and cell death. However, their molecular triggers and the relative timing of these events have not been fully elucidated. Here, we report that DSBs accumulate relatively early after UV irradiation in pol η-depleted cells. Despite the availability of repair pathways, DSBs persist and chromosome instability (CIN) is not detectable. Later on cells with pan-nuclear γH2AX and massive exposure of template single-stranded DNA (ssDNA), which indicate severe replication stress, accumulate and such events are followed by cell death. Reinforcing the causal link between the accumulation of pan-nuclear ssDNA/γH2AX signals and cell death, downregulation of RPA increased both replication stress and the cell death of pol η-deficient cells. Remarkably, DSBs, pan-nuclear ssDNA/γH2AX, S-phase arrest, and cell death are all attenuated by MRE11 nuclease knockdown. Such results suggest that unscheduled MRE11-dependent activities at replicating DNA selectively trigger cell death, but not CIN. Together these results show that pol η-depletion promotes a type of cell death that may be attractive as a therapeutic tool because of the lack of CIN.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , ADN Polimerasa Dirigida por ADN/genética , Histonas/genética , Proteína Homóloga de MRE11/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Muerte Celular/genética , Inestabilidad Cromosómica/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , Humanos , Fase S/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.
Asunto(s)
Reparación del ADN/genética , Replicación del ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Cromátides/genética , Cromátides/efectos de la radiación , Inestabilidad Cromosómica/efectos de la radiación , Cromosomas/genética , Cromosomas/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Anemia de Fanconi/patología , Inestabilidad Genómica/genética , Inestabilidad Genómica/efectos de la radiación , Humanos , ARN Interferente Pequeño , Rayos UltravioletaRESUMEN
Ataxia telangiectasia (AT) is a rare neurodegenerative disorder, inherited in an autosomal recessive manner. Total blood samples were collected from 20 patients with AT, 13 parents of patients, and 17 healthy volunteers. This study aimed at evaluating the frequency of chromosomal breaks in spontaneous cultures, induced by bleomycin and ionizing radiation, and further evaluated the rates of oxidative stress in AT patients and in their parents, compared to a control group. Three cell cultures were performed to each individual: the first culture did not receive induction to chromosomal instability, the second was exposed to bleomycin, and the last culture was exposed to ionizing radiation. To evaluate the rates of oxidative stress, the markers superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid (TBARS) were utilized. Significant differences were observed between the three kinds of culture treatments (spontaneous, bleomycin, and radiation induced) and the breaks and chromosomal aberrations in the different groups. The oxidative stress showed no significant differences between the markers. This study showed that techniques of chromosomal instability after the induction of ionizing radiation and bleomycin are efficient in the identification of syndrome patients, with the ionizing radiation being the most effective.
Asunto(s)
Ataxia Telangiectasia/genética , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Adulto , Ataxia Telangiectasia/patología , Bleomicina/farmacología , Células Cultivadas , Inestabilidad Cromosómica/genética , Aberraciones Cromosómicas/efectos de los fármacos , Aberraciones Cromosómicas/efectos de la radiación , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/genética , Linaje , Radiación IonizanteRESUMEN
The aim of this study was to investigate whether there is a differential response of lymphocytes from healthy MnSOD genotype subjects to oxidative stress. We used UV radiation as a toxic agent due to its genotoxic effects associated with chromosome aberrations caused by breaks in the DNA strands. Cellular growth rate, cell viability, mitotic index, chromosomal instability and biomarkers of oxidative metabolism were analysed in lymphocyte cells from healthy adults with different Ala16Val MnSOD polymorphisms that produce tree genotypes: AA, VV and AV. We found a differential response to UV exposure in cultures of lymphocyte cells from Ala16Val genotype donors. In general, AA cell cultures presented higher viability and mitotic index and lower TBARS levels than VV and AV cells for both the control and UV exposure groups. However, when we compared the DNA damage among the three genotypes, AA lymphocyte cells presented the highest damage from UV exposure. These data suggest that the Ala16Val polymorphism affects the response of cellular oxidative metabolism in different ways.