RESUMO
BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sítios de Ligação , Cromatografia em Gel , DNA Nucleotidilexotransferase/genética , Regulação para Baixo , Deleção de Genes , Regulação da Expressão Gênica , Biblioteca Gênica , Genes BRCA1/fisiologia , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/genética , Recombinação Genética , Fatores de Transcrição/química , Células Tumorais Cultivadas/enzimologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.
Assuntos
Proteínas de Transporte/isolamento & purificação , DNA Nucleotidilexotransferase/metabolismo , NF-kappa B , Proteínas Nucleares/isolamento & purificação , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Mapeamento Cromossômico , DNA Nucleotidilexotransferase/genética , Proteínas de Ligação a DNA , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Transcrição RelA , Técnicas do Sistema de Duplo-HíbridoRESUMO
The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.
Assuntos
Caspases/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Northern Blotting , Western Blotting , Inibidores de Caspase , Sobrevivência Celular , Meios de Cultura Livres de Soro , Etoposídeo/farmacologia , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Dedos de ZincoRESUMO
We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.
