RESUMEN
The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn(2+)-dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hook-dependent Mre11 complex functions. One of these alleles, Rad50(46), conferred reduced Zn(2+) affinity and dimerization efficiency. Homozygous Rad50(46/46) mutations were lethal in mice. However, in the presence of wild-type Rad50, Rad50(46) exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50(+/46) exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50(+/46) Atm(+/-) mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Daño del ADN , Transducción de Señal/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carcinogénesis/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Germinativas/patología , Proteína Homóloga de MRE11 , Ratones , Mutación , Fenotipo , Estructura Terciaria de ProteínaRESUMEN
The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11 (ATLD1) allele. Mre11 (ATLD1/ATLD1) females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1 (-/-) and Trp13 (Gt/Gt) mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11 (ATLD1/ATLD1) . Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11 (ATLD1/ATLD1) follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.
Asunto(s)
Emparejamiento Cromosómico , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/fisiología , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Meiosis , Oocitos/metabolismo , Oogonios/metabolismo , Animales , ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Proteína Homóloga de MRE11 , Ratones , Ratones Transgénicos , Oogénesis , Oogonios/fisiologíaRESUMEN
Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation in lymphoid cells is controlled by a recently identified protein, cyclin O, and its activity is modulated by p53 and members of the Bcl-2 protein family. In this chapter, we describe methods for measuring changes in Cdk2 activity during apoptosis. In addition, we also show the details of the generation of an antibody able to immunoprecipitate the cyclin O complexes from apoptotic cells in native conditions and its use to measure the kinase activity associated with this proapoptotic cyclin.
Asunto(s)
Apoptosis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inmunoprecipitación/métodos , Animales , Ciclo Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/análisis , Ciclinas/inmunología , Ciclinas/metabolismo , Humanos , Linfocitos T/citologíaRESUMEN
B-cell lymphoma-2 (Bcl-2) family members have been demonstrated to play a crucial role in the regulation of apoptosis as mediators in between the apical stimuli sensing steps and the executory mechanisms of apoptosis. Deregulation of their role may subvert the homeostasis of a given tissue and collaborate in the genesis of a myriad of diseases characterised by exacerbated or insufficient apoptosis, including diseases such as neurodegenerative diseases or cancer. Structural studies have defined homology regions shared by the members of the family that are responsible of the network of interactions established amongst the members of the family. These proteins usually form heterodimers between the so called antiapoptotic multidomain members and the proapoptotic BH3-only proteins. As a consequence, mitochondrial apoptogenic proteins are released to the cytoplasm and the apoptotic signal proceeds towards the final, execution phase of the apoptotic process. The high complexity of the family (more than 20 members have been isolated) makes the study of individual proteins difficult. Genetic approaches have revealed a high degree of redundancy in the family. Only a few proteins belonging to the antiapoptotic group have been proven to be essential for correct embryonic development. Genetic inactivation in mice shows a dramatic phenotype characterised by massive cell death in multiple tissues during embryogenesis, which leads from very early up to perinatal death. This genetic evidence proves the importance of the members of the family for the regulation of apoptosis in order to achieve the proper development and homeostasis of tissues and organs.
Asunto(s)
Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Ratones , Ratones Noqueados , Modelos Teóricos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.