RESUMO
Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double-strand break (DSB)-induced extensive LOH, and its suppression, using a non-essential minichromosome, Ch(16), in fission yeast. We find extensive LOH to arise from a new break-induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1-dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break-induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome-wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.
Assuntos
Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Conversão Gênica , Perda de Heterozigosidade , Schizosaccharomyces/genética , Adenosina Trifosfatases/metabolismo , Centrômero/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.
Assuntos
Quebras de DNA de Cadeia Dupla , Perda de Heterozigosidade/genética , Recombinação Genética , Schizosaccharomyces/genética , Telômero/metabolismo , Translocação Genética , Alelos , Sequência de Bases , Cromossomos Fúngicos/metabolismo , Troca Genética , Reparo do DNA , Marcadores Genéticos , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Mutação/genética , Filogenia , Rad51 Recombinase/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
In rapidly dividing bacterial cells, the machinery for repair of DNA double-strand breaks has to contend not only with the forces driving replication and transmission of the DNA but also its transcription. By exploiting I-SceI homing endonuclease to break the Escherichia coli chromosome at one or more defined locations, we have been able to investigate how these processes are co-ordinated and repair is accomplished. When breaks are induced at a single site, the SOS-inducible RecN protein and the transcription factor DksA combine to promote efficient repair. When induced at two or more, distantly located sites, RecN becomes almost indispensable. Many cells that do survive have extensive deletions of sequences flanking the break, with end points often coinciding with imperfect repeat elements. These findings herald a much greater complexity for chromosome repair than suggested by current mechanistic models and reveal a role for RecN in protecting the chromosome from break-induced chromosome rearrangements.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA/fisiologia , Enzimas de Restrição do DNA/metabolismo , DNA/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Pareamento de Bases , Sequência de Bases , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , Enzimas de Restrição do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/genética , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Dados de Sequência Molecular , Mutação , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Recombinação Genética , Resposta SOS em Genética/genética , Proteínas de Saccharomyces cerevisiae , Deleção de Sequência , Fatores de Transcrição/genéticaRESUMO
Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.