RESUMO
DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.
Assuntos
Apoptose/fisiologia , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes myc/fisiologia , Proteínas HMGB/metabolismo , Regiões de Interação com a Matriz/fisiologia , Neurônios/citologia , Fatores de Transcrição/metabolismo , Apoptose/genética , Sequência de Bases , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Genes myc/genética , Proteínas HMGB/genética , Humanos , Células Jurkat , Regiões de Interação com a Matriz/genética , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genéticaRESUMO
DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.
