RESUMO
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Transporte/genética , DNA de Helmintos/genética , Proteínas de Ligação a DNA/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Helmintos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Imagem Individual de MoléculaRESUMO
Cohesin and condensin complexes are essential for defining the topology of chromosomes through the cell cycle. Here we look at the emerging role of these complexes in regulating chromatin structure and gene expression and reflect on how these activities could be linked with chromosome topology.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Eucariotos , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Estruturas Cromossômicas , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Eucariotos/citologia , Eucariotos/genética , Regulação da Expressão Gênica , Humanos , Mitose/genética , Complexos Multiproteicos/genética , CoesinasRESUMO
BACKGROUND: Chromosome segregation and the repair of DNA double-strand breaks (DSBs) by homologous recombination require cohesin, the protein complex that mediates sister chromatid cohesion (SCC). In addition, cohesin is also required for the integrity of DNA damage checkpoints in somatic cells, where cohesin loading depends on a conserved complex containing the Scc2/Nipbl protein. Although cohesin is required for the completion of meiotic recombination, little is known about how cohesin promotes the repair of meiotic DSBs and about the factors that promote loading of cohesin during meiosis. RESULTS: Here we show that during Caenorhabditis elegans meiosis, loading of cohesin requires SCC-2, whereas the cohesin-related complexes condensin and SMC-5/6 can be loaded by mechanisms independent of both SCC-2 and cohesin. Although the lack of cohesin in scc-2 mutants impairs the repair of meiotic DSBs, surprisingly, the persistent DNA damage fails to trigger an apoptotic response of the conserved pachytene DNA damage checkpoint. Mutants carrying an scc-3 allele that abrogates loading of meiotic cohesin are also deficient in the apoptotic response of the pachytene checkpoint, and both scc-2 and scc-3 mutants fail to recruit the DNA damage sensor 9-1-1 complex onto persistent damage sites during meiosis. Furthermore, we show that meiotic cohesin is also required for the timely loading of the RAD-51 recombinase to irradiation-induced DSBs. CONCLUSIONS: We propose that meiotic cohesin promotes DSB processing and recruitment of DNA damage checkpoint proteins, thus implicating cohesin in the earliest steps of the DNA damage response during meiosis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Meiose , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Estágio Paquíteno , CoesinasRESUMO
Fanconi anemia (FA) is a complex cancer susceptibility disorder associated with DNA repair defects and infertility, yet the precise function of the FA proteins in genome maintenance remains unclear. Here we report that C. elegans FANCD2 (fcd-2) is dispensable for normal meiotic recombination but is required in crossover defective mutants to prevent illegitimate repair of meiotic breaks by nonhomologous end joining (NHEJ). In mitotic cells, we show that DNA repair defects of C. elegans fcd-2 mutants and FA-deficient human cells are significantly suppressed by eliminating NHEJ. Moreover, NHEJ factors are inappropriately recruited to sites of replication stress in the absence of FANCD2. Our findings are consistent with the interpretation that FA results from the promiscuous action of NHEJ during DNA repair. We propose that a critical function of the FA pathway is to channel lesions into accurate, as opposed to error-prone, repair pathways.
Assuntos
Reparo do DNA/genética , Anemia de Fanconi/genética , Recombinação Genética , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Troca Genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Meiose/genética , Mutação/genética , Rad51 Recombinase/metabolismo , Estresse FisiológicoRESUMO
Hexaploid wheat possesses 42 chromosomes derived from its three ancestral genomes. The 21 pairs of chromosomes can be further divided into seven groups of six chromosomes (one chromosome pair being derived from each of the three ancestral genomes), based on the similarity of their gene order. Previous studies have revealed that, during anther development, the chromosomes associate in 21 pairs via their centromeres. The present study reveals that, as a prelude to meiosis, these 21 chromosome pairs in hexaploid (and tetraploid) wheat associate via the centromeres into seven groups as the telomeres begin to cluster. This results in the association of multiple chromosomes, which then need to be resolved as meiosis progresses. The formation of the seven chromosome clusters now explains the occasional occurrence of remnants of multiple associations, which have been reported at later stages of meiosis in hexaploid (and tetraploid) wheat. Importantly, the chromosomes have the opportunity to be resorted via these multiple interactions. As meiosis progresses, such interactions are resolved through the action of loci such as Ph1, leaving chromosomes as homologous pairs.