RESUMEN
Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.
Asunto(s)
Roturas del ADN de Doble Cadena , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos Nucleares/metabolismo , Southern Blotting , Inmunoprecipitación de Cromatina , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Fúngicos , Autoantígeno Ku , Complejos Multiproteicos/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factores de TiempoRESUMEN
In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Ligasas/metabolismo , Reparación del ADN , ADN/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Daño del ADN , ADN Ligasas/química , ADN Ligasas/genética , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Exonucleasas/química , Exonucleasas/metabolismo , Mutación , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Recombinación Genética , Saccharomyces cerevisiae/genéticaRESUMEN
The repair of DNA double-strand breaks by non-homologous end-joining (NHEJ) has long been thought to be restricted to eukaryotes. However, recent papers document the existence of operons encoding functional NHEJ complexes in some bacteria. These findings provide new evolutionary insights into the core biochemistry of this repair pathway, and suggest that one function driving the selection of NHEJ in bacteria, and perhaps eukaryotes, relates to prolonged periods of mitotic exit.
Asunto(s)
Bacterias/genética , Cromosomas Bacterianos/genética , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Bacterias/metabolismo , ADN Ligasas/genética , Humanos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.