RESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.
Assuntos
Quebra Cromossômica , Reparo do DNA , DNA/análise , Reação em Cadeia da Polimerase/métodos , Translocação Genética , Células Cultivadas , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Dano ao DNA , Primers do DNA/química , HumanosRESUMO
Activation of apoptosis introduces a site-specific break within intron 11 of the MLL gene. Using the CD95 apoptotic signaling pathway in human lymphoblastoid cells, the 5' fragment of MLL undergoes translocation to intron 4 of AF9 and the proleukemogenic MLL-AF9 fusion gene created is transcribed. Both the breaks in MLL and transcription of the MLL-AF9 fusion gene are suppressed in the presence of the broad spectrum caspase inhibitor, zVAD.fmk. Duplicate cells containing sequence identical MLL-AF9 fusion junctions were identified within a cell population that had recovered from apoptosis. This indicated that cells harboring a translocation initiated by apoptotic cleavage had divided. These data are consistent with a novel pathogenic role for the apoptotic program where translocations with leukemogenic potential are created within cells that have the capacity to divide.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Divisão Celular/genética , Linhagem Celular , Quebra Cromossômica , Proteínas de Ligação a DNA/biossíntese , Histona-Lisina N-Metiltransferase , Humanos , Íntrons , Linfócitos/citologia , Linfócitos/metabolismo , Linfócitos/fisiologia , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/métodos , Transdução de Sinais/fisiologia , Transcrição Gênica , Receptor fas/fisiologiaRESUMO
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.
Assuntos
Quebra Cromossômica/genética , Dano ao DNA , Reação em Cadeia da Polimerase/métodos , Translocação Genética/genética , Animais , DNA/análise , DNA/genética , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Proteína de Leucina Linfoide-Mieloide , Proto-Oncogenes/genética , Fatores de Transcrição/genéticaRESUMO
The pathological consequences resulting from deregulation of the apoptotic program include cancer (too little apoptosis) or diseases of cell deprivation, such as Alzheimer's (too much apoptosis). We have identified an additional pathology whereby cells reaching the earliest stage of chromatin cleavage have the potential to suppress apoptotic execution and survive. One specific cleavage event associated with this process is restricted to a location within the mixed lineage leukemia (MLL) gene at 11q23. The site of cleavage is consistent with the location where large, approximately 50 kbp loops of supercoiled DNA are attached to the nuclear matrix. Cells modified by this process generate MLL translocations, as shown by inverse PCR, that survive for days to weeks but which have no known relationship with clinical disease. Using a specific approach, cells stimulated by anti-CD95 antibody, a potent stimulator of the apoptotic program, facilitated creation of the MLL-AF9 fusion gene. Further, this rearrangement, which is commonly observed in patients with AML linked to exposure to cytotoxic agents, was efficiently transcribed in cells that were able to undergo cell division. These data are discussed in the context of benzene and benzene metabolite toxicity that impacts the process of apoptosis and is known to lead to leukemic disease.