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
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
We present a simple and efficient method for controlled linear induction of DNA damage in live cells. By passing a pulsed laser beam through a cylindrical lens prior to expansion, an elongated elliptical beam profile is created with the ability to expose controlled linear patterns while keeping the beam and the sample stationary. The length and orientation of the beam at the sample plane were reliably controlled by an adjustable aperture and rotation of the cylindrical lens, respectively. Localized immunostaining by the DNA double strand break (DSB) markers phosphorylated H2AX (gamma H2AX) and Nbs1 in the nuclei of HeLa cells exposed to the "line scissors" was shown via confocal imaging. The line scissors method proved more efficient than the scanning mirror and scanning stage methods at induction of DNA DSB damage with the added benefit of having a greater potential for high throughput applications.