RESUMEN
Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN/genética , Heterocromatina/genética , Recombinación Homóloga/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA2/genética , Células CHO , Cricetulus , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteínas Represoras/genética , Proteína 28 que Contiene Motivos TripartitoRESUMEN
DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Fase G2 , Línea Celular , Heterocromatina/metabolismo , Humanos , Cinética , Redes y Vías Metabólicas , Recombinación GenéticaRESUMEN
DNA double-strand breaks (DSBs) represent the most biologically significant lesions induced by ionizing radiation (IR). HR is the predominant pathway for repairing one-ended DSBs arising in S-phase when the replication fork encounters single-stranded breaks or base damages. Here, we discuss recent findings that two-ended DSBs directly induced by X- or γ-rays in late S- or G2-phase are repaired predominantly by NHEJ, with HR only repairing a sub-fraction of such DSBs. This sub-fraction represents DSBs which localize to heterochromatic DNA regions and, which in control cells, are repaired with slow kinetics over many hours post irradiation. The observation that defined DSB populations are repaired by either NHEJ or HR suggests an assignment of specific tasks for each of the two processes. Furthermore, heavy ion induced complex DSBs, which are in general more slowly repaired than X- or γ-ray induced breaks, are nearly always repaired by HR independent of chromatin localization suggesting that the speed of repair is an important factor determining the DSB repair pathway usage. Finally, NHEJ and HR can, under certain conditions, also compensate for each other such that DSBs normally repaired by one pathway can undergo repair by the other if genetic failures necessitate the pathway switch.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Recombinación Homóloga/genética , Radiación Ionizante , Fase G2/genética , Fase G2/efectos de la radiación , Marcación de Gen/métodos , HumanosRESUMEN
The MUS81 endonuclease complex has been shown to play an important role in the repair of stalled or blocked replication forks and in the processing of meiotic recombination intermediates from yeast to humans. This endonuclease is composed of two subunits, MUS81 and EME1. Surprisingly, unlike other organisms, Arabidopsis (Arabidopsis thaliana) has two EME1 homologs encoded in its genome. AtEME1A and AtEME1B show 63% identity on the protein level. We were able to demonstrate that, after expression in Escherichia coli, each EME1 protein can assemble with the unique AtMUS81 to form a functional endonuclease. Both complexes, AtMUS81-AtEME1A and AtMUS81-AtEME1B, are not only able to cleave 3'-flap structures and nicked Holliday junctions (HJs) but also, with reduced efficiency, intact HJs. While the complexes have the same cleavage patterns with both nicked DNA substrates, slight differences in the processing of intact HJs can be detected. Our results are in line with an involvement of both MUS81-EME1 endonuclease complexes in DNA recombination and repair processes in Arabidopsis.