RESUMEN
Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.
Asunto(s)
Daño del ADN , Rayos Láser , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Células HeLa , Histonas/análisis , Humanos , Láseres de Colorantes , Rayos UltravioletaRESUMEN
Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.
Asunto(s)
Cromosomas/química , Cromosomas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , ADN/genética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interfase , Cinesinas/metabolismo , Sustancias Macromoleculares , Mitosis , Complejos Multiproteicos , Pruebas de Precipitina , Unión Proteica , Transporte de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Xenopus , ADN Metiltransferasa 3BRESUMEN
Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Reparación del ADN , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Interferencia de ARNRESUMEN
A proper response to DNA damage is critical for the maintenance of genome integrity. However, it is difficult to study the in vivo kinetics and factor requirements of the damage recognition process in mammalian cells. In order to address how the cell reacts to DNA damage, we utilized a second harmonic (532 nm) pulsed Nd:YAG laser to induce highly concentrated damage in a small area in interphase cell nuclei and cytologically analyzed both protein recruitment and modification. Our results revealed for the first time the sequential recruitment of factors involved in two major DNA double-strand break (DSB) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), and the cell cycle-specific recruitment of the sister chromatid cohesion complex cohesin to the damage site. In this chapter, the strategy developed to study the DNA damage response using the 532-nm Nd:YAG laser will be summarized.
Asunto(s)
Daño del ADN , Reparación del ADN , Rayos Láser , Animales , Cafeína/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Inmunoprecipitación de Cromatina , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Células HeLa , Humanos , Imagenología Tridimensional , Radiación IonizanteRESUMEN
Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.