RESUMEN
Nearly every mammalian cell division is accompanied by a mutational event that becomes fixed in a daughter cell. When carried forward to additional cell progeny, a clone of variant cells can emerge. As a result, mammals are complex mosaics of clones that are genetically distinct from one another. Recent high-throughput sequencing studies have revealed that mosaicism is common, clone sizes often increase with age and specific variants can affect tissue function and disease development. Variants that are acquired during early embryogenesis are shared by multiple cell types and can affect numerous tissues. Within tissues, variant clones compete, which can result in their expansion or elimination. Embryonic mosaicism has clinical implications for genetic disease severity and transmission but is likely an under-recognized phenomenon. To better understand its implications for mosaic individuals, it is essential to leverage research tools that can elucidate the mechanisms by which expanded embryonic variants influence development and disease.
RESUMEN
Homologous recombination (HR) is a template-driven repair pathway that mends DNA double-stranded breaks (DSBs), and thus helps to maintain genome stability. The RAD51 recombinase facilitates DNA joint formation during HR, but to accomplish this task, RAD51 must be loaded onto the single-stranded DNA. DSS1, a candidate gene for split hand/split foot syndrome, provides the ability to recognize RPA-coated ssDNA to the tumor suppressor BRCA2, which is complexed with RAD51. Together BRCA2-DSS1 displace RPA and load RAD51 onto the ssDNA. In addition, the BRCA2 interacting protein BCCIP normally colocalizes with chromatin bound BRCA2, and upon DSB induction, RAD51 colocalizes with BRCA2-BCCIP foci. Down-regulation of BCCIP reduces DSB repair and disrupts BRCA2 and RAD51 foci formation. While BCCIP is known to interact with BRCA2, the relationship between BCCIP and RAD51 is not known. In this study, we investigated the biochemical role of the ß-isoform of BCCIP in relation to the RAD51 recombinase. We demonstrate that BCCIPß binds DNA and physically and functionally interacts with RAD51 to stimulate its homologous DNA pairing activity. Notably, this stimulatory effect is not the result of RAD51 nucleoprotein filament stabilization; rather, we demonstrate that BCCIPß induces a conformational change within the RAD51 filament that promotes release of ADP to help maintain an active presynaptic filament. Our findings reveal a functional role for BCCIPß as a RAD51 accessory factor in HR.
Asunto(s)
Adenosina Difosfato/metabolismo , Emparejamiento Base , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Recombinación Homóloga , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Ciclo Celular/química , Reparación del ADN , Humanos , Hidrólisis , Proteínas Nucleares/química , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Multimerización de ProteínaRESUMEN
The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacologic target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate the protective role of SOD1 against oxidative stress in PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.
RESUMEN
The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.
Asunto(s)
Proteína Fosfatasa 2C , Superóxido Dismutasa-1 , Proteína Fosfatasa 2C/metabolismo , Proteína Fosfatasa 2C/genética , Humanos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Línea Celular Tumoral , Leucemia/genética , Sistemas CRISPR-Cas , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Mutaciones Letales Sintéticas , MutaciónRESUMEN
During aging, blood cell production becomes dominated by a limited number of variant hematopoietic stem cell (HSC) clones. Differentiated progeny of variant HSCs are thought to mediate the detrimental effects of such clonal hematopoiesis on organismal health, but the mechanisms are poorly understood. While somatic mutations in DNA methyltransferase 3A (DNMT3A) frequently drive clonal dominance, the aging milieu also likely contributes. Here, we examined in mice the interaction between high-fat diet (HFD) and reduced DNMT3A in hematopoietic cells; strikingly, this combination led to weight gain. HFD amplified pro-inflammatory pathways and upregulated inflammation-associated genes in mutant cells along a pro-myeloid trajectory. Aberrant DNA methylation during myeloid differentiation and in response to HFD led to pro-inflammatory activation and maintenance of stemness genes. These findings suggest that reduced DNMT3A in hematopoietic cells contributes to weight gain, inflammation, and metabolic dysfunction, highlighting a role for DNMT3A loss in the development of metabolic disorders.
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[This corrects the article DOI: 10.1016/j.isci.2024.109122.].
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Many solid cancers metastasize to the bone and bone marrow (BM). This process may occur even before the diagnosis of primary tumors, as evidenced by the discovery of disseminated tumor cells (DTCs) in patients without occult malignancies. The cellular fates and metastatic progression of DTCs are determined by complicated interactions between cancer cells and BM niches. Not surprisingly, these niches also play important roles in normal biology, including homeostasis and turnover of skeletal and hematopoiesis systems. In this Review, we summarize recent findings on functions of BM niches in bone metastasis (BoMet), particularly during the early stage of colonization. In light of the rich knowledge of hematopoiesis and osteogenesis, we highlight how DTCs may progress into overt BoMet by taking advantage of niche cells and their activities in tissue turnover, especially those related to immunomodulation and bone repair.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Médula Ósea/inmunología , Neoplasias de la Médula Ósea/patología , Neoplasias de la Médula Ósea/secundario , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Remodelación Ósea/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Privilegio Inmunológico , Tolerancia Inmunológica , Masculino , Modelos Biológicos , Células Mieloides/inmunología , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/terapia , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Nicho de Células Madre/inmunología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
The meiosis-specific recombinase, DMC1, is important for the generation of haploids during meiosis. DMC1 forms a helical nucleoprotein filament on ssDNA overhangs located at the processed double-stranded DNA break. The DMC1 filament performs a search for homology in homologous chromosome. Once homology is located, the DMC1 filament strand invades the homologous chromosome forming a displacement loop (D-loop). These connections are needed for accurate segregation to occur later in meiosis. Because DMC1 requires numerous accessory factors and specific ionic conditions to participate in this DNA repair process, in vitro assays were developed to understand how these accessory factors influence the biochemical properties of hDMC1. This chapter describes a method that can be used to investigate the stability of the human DMC1 nucleoprotein filament under various conditions and provides insight into an important early stage in DNA double-strand break repair by homologous recombination during meiosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/metabolismo , Recombinasas/metabolismo , Reparación del ADN por Recombinación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Meiosis/genética , Nucleoproteínas/genética , Nucleoproteínas/aislamiento & purificación , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Recombinasas/genética , Recombinasas/aislamiento & purificaciónRESUMEN
Meiosis is a specialized cell division that generates gametes. Meiotic recombination is essential not only to generate diversity in offspring, but also to hold homologous chromosomes together through chiasma allowing proper chromosome segregation. This process requires the meiosis-specific recombinase, DMC1. DMC1 facilitates the search for homology between the homologous chromosomes and is followed by DNA strand invasion and strand exchange to produce a linkage between the two homologous chromosomes. The development of biochemical in vitro assays and the purification of the requisite proteins factors has led to a better understanding of the molecular mechanisms of meiotic homologous recombination. In this chapter, a detailed in vitro assay to examine DNA strand exchange over 5000 bases of DNA catalyzed by human DMC1 is described. This method has proved to be valuable for examining the catalytic potential of hDMC1 and delineating the functional interaction with other HR factors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Pruebas de Enzimas/métodos , Plásmidos/metabolismo , Recombinasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , ADN/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Meiosis , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Recombinasas/genética , Recombinasas/aislamiento & purificación , Reparación del ADN por RecombinaciónRESUMEN
Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity.