RESUMO
DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.
Assuntos
Núcleo Celular/metabolismo , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/química , DNA Ligases/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Humanos , Linfócitos/metabolismo , Sinais de Localização NuclearRESUMO
The pathway of V(D)J recombination was discovered almost three decades ago. Yet it continues to baffle scientists because of its inherent complexity and the multiple layers of regulation that are required to efficiently generate a diverse repertoire of T and B cells. The non-homologous end-joining (NHEJ) DNA repair pathway is an integral part of the V(D)J reaction, and its numerous players perform critical functions in generating this vast diversity, while ensuring genomic stability. In this review, we summarize the efforts of a number of laboratories including ours in providing the mechanisms of V(D)J regulation with a focus on the NHEJ pathway. This involves discovering new players, unraveling unknown roles for known components, and understanding how deregulation of these pathways contributes to generation of primary immunodeficiencies. A long-standing interest of our laboratory has been to elucidate various mechanisms that control RAG activity. Our recent work has focused on understanding the multiple protein-protein interactions and protein-DNA interactions during V(D)J recombination, which allow efficient and regulated generation of the antigen receptors. Exploring how deregulation of this process contributes to immunodeficiencies also continues to be an important area of research for our group.
Assuntos
Reparo do DNA por Junção de Extremidades , Recombinação V(D)J , Animais , Humanos , Modelos Moleculares , Imunodeficiência Combinada Severa/genéticaRESUMO
Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis-Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA-protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.
Assuntos
DNA Ligases/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Recombinação V(D)J/fisiologia , Sequência de Aminoácidos , Sequência de Bases , DNA Ligase Dependente de ATP , Primers do DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Polarização de Fluorescência , Vetores Genéticos/genética , Células HeLa , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosforilação , Mutação Puntual/genética , Análise de Sequência de DNA , TransfecçãoRESUMO
Cancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.
