RESUMO
The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.
Assuntos
Dano ao DNA , Lamina Tipo A , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Fosforilação , Nucleotidiltransferases/genética , DNA/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Chk1 is a member of the DNA damage response pathway, whose loss leads to replication stress and genome instability. Because of its protective role against lethal levels of DNA replication stress, Chk1 has been studied as a valuable and intriguing target for cancer therapy. However, one of the most prominent challenges with this strategy is development of resistance to Chk1 inhibitors, rendering the treatment ineffective. In their recent papers, Hunter and colleagues demonstrate multiple mechanisms by which Chk1 inhibitor resistance can arise in lymphomas. Specifically, this series of papers identify the relationship between dysfunction in NF-κB and the development of Chk1 inhibitor resistance through a loss of Chk1 activity in mouse models of lymphoma. They identify that cells lacking Chk1 activity can compensate for this loss through up-regulation of alternative pathways, such as PI3K/AKT. Finally, this work also identifies a novel role for Claspin, an important Chk1 activator, in female fertility and cancer development, furthering our understanding of how dysfunction in the Claspin/Chk1 signaling pathway affects disease states. These findings improve our understanding of drug resistance in cancer therapy, which has important implications for clinical use of Chk1 inhibitors.
Assuntos
Linfoma , Proteínas Quinases , Feminino , Camundongos , Animais , Quinase 1 do Ponto de Checagem/genética , Proteínas Quinases/metabolismo , Fosfatidilinositol 3-Quinases , Recidiva Local de Neoplasia , Linfoma/tratamento farmacológico , Linfoma/genética , Inibidores de Proteínas QuinasesRESUMO
Polo-like kinase 1 (PLK1) protects against genome instability by ensuring timely and accurate mitotic cell division. PLK1 activity is tightly regulated throughout the cell cycle. Although the pathways that initially activate PLK1 in G2 are well-characterized, the factors that directly regulate PLK1 in mitosis remain poorly understood. Here, we identify that human PLK1 activity is sustained by the DNA damage response kinase Checkpoint kinase 2 (Chk2) in mitosis. Chk2 directly phosphorylates PLK1 T210, a residue on its T-loop whose phosphorylation is essential for full PLK1 kinase activity. Loss of Chk2-dependent PLK1 activity causes increased mitotic errors, including chromosome misalignment, chromosome missegregation, and cytokinetic defects. Moreover, Chk2 deficiency increases sensitivity to PLK1 inhibitors, suggesting that Chk2 status may be an informative biomarker for PLK1 inhibitor efficacy. This work demonstrates that Chk2 sustains mitotic PLK1 activity and protects genome stability through discrete functions in interphase DNA damage repair and mitotic chromosome segregation.
RESUMO
Centromere protein A (CENP-A) defines centromere identity and nucleates kinetochore formation for mitotic chromosome segregation. Here, we show that ataxia telangiectasia and Rad3-related (ATR) kinase, a master regulator of the DNA damage response, protects CENP-A occupancy at interphase centromeres in a DNA damage-independent manner. In unperturbed cells, ATR localizes to promyelocytic leukemia nuclear bodies (PML NBs), which house the histone H3.3 chaperone DAXX (death domain-associated protein 6). We find that ATR inhibition reduces DAXX association with PML NBs, resulting in the DAXX-dependent loss of CENP-A and an aberrant increase in H3.3 at interphase centromeres. Additionally, we show that ATR-dependent phosphorylation within the C terminus of DAXX regulates CENP-A occupancy at centromeres and DAXX localization. Lastly, we demonstrate that acute ATR inhibition during interphase leads to kinetochore formation defects and an increased rate of lagging chromosomes. These findings highlight a mechanism by which ATR protects centromere identity and genome stability.
Assuntos
Centrômero , Corpos Nucleares da Leucemia Promielocítica , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
Maintenance of an intact genome is essential for cellular and organismal homeostasis. The centromere is a specialized chromosomal locus required for faithful genome inheritance at each round of cell division. Human centromeres are composed of large tandem arrays of repetitive alpha-satellite DNA, which are often sites of aberrant rearrangements that may lead to chromosome fusions and genetic abnormalities. While the centromere has an essential role in chromosome segregation during mitosis, the long and repetitive nature of the highly identical repeats has greatly hindered in-depth genetic studies, and complete annotation of all human centromeres is still lacking. Here, we review our current understanding of human centromere genetics and epigenetics as well as recent investigations into the role of centromere DNA in disease, with a special focus on cancer, aging, and human immunodeficiencyâ»centromeric instabilityâ»facial anomalies (ICF) syndrome. We also highlight the causes and consequences of genomic instability at these large repetitive arrays and describe the possible sources of centromere fragility. The novel connection between alpha-satellite DNA instability and human pathological conditions emphasizes the importance of obtaining a truly complete human genome assembly and accelerating our understanding of centromere repeats' role in physiology and beyond.
RESUMO
A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. The N gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates. Additionally, it was found to contain regions specific to each genotype conducive to probe design. The RT-PCR assay described amplifies a portion of the nucleoprotein gene of all 107 rabies and rabies-related viruses, but none of the other viruses tested. Inclusion of TaqMan-genotype-specific probes in the RT-PCR assay permits rapid identification of the virus present. By combining RT-PCR with TaqMan genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination.