RESUMEN
Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Fase G2/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , ADN Helicasas , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas , Fibroblastos/efectos de la radiación , Fase G1/efectos de la radiación , Eliminación de Gen , Células HeLa , Heterocromatina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Proteínas Portadoras/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Etopósido/toxicidad , Proteínas Nucleares/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Endodesoxirribonucleasas , Fase G1/efectos de los fármacos , Fase G1/genética , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Treonina/metabolismoRESUMEN
DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain.
Asunto(s)
Rotura Cromosómica , Roturas del ADN de Doble Cadena , Reparación del ADN , Fase G2/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Endonucleasas , Humanos , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: About 5-10% of all breast cancer cases are associated with heterozygous germ-line mutations in the genes encoding BRCA1 and BRCA2. Carriers of such mutations are highly predisposed for developing breast or ovarian cancer and, thus, are advised to undergo regular radio-diagnostic examinations. BRCA1 and BRCA2 are involved in multiple cellular processes including the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) and different studies addressing the DSB repair capacity of BRCA1+/- or BRCA2+/- cells led to contradictory results. MATERIALS AND METHODS: Using the sensitive method of γH2AX foci analysis in combination with cell cycle markers, we specifically measured DSB repair in confluent G0 as well as in exponentially growing G1 and G2 phase primary WT, BRCA1+/- and BRCA2+/- fibroblasts. RESULTS: Both BRCA1+/- and BRCA2+/- cells displayed normal DSB repair in G0 and in G1. In contrast, in G2, BRCA2+/- but not BRCA1+/- cells exhibited a decreased DSB repair capacity which was in between that of WT and that of a hypomorphic BRCA2-/- cell line. CONCLUSIONS: The residual amount of normal BRCA1 seems to be sufficient for efficient DSB repair in all cell cycle phases, while the decreased DSB repair capacity of heterozygous BRCA2 mutations suggests gene dosage effects in G2.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Fibroblastos/efectos de la radiación , Fase G2/efectos de la radiación , Histonas/efectos de la radiación , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral/efectos de la radiación , Femenino , Fibroblastos/patología , Heterocigoto , Histonas/análisis , Humanos , Dosis de Radiación , Tolerancia a Radiación , Radiación IonizanteRESUMEN
DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating gammaH2AX foci that can be visualized by immunofluorescence. There is a close correlation between gammaH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, gammaH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionizing radiation, gammaH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimize the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.