RESUMEN
The DNA damage response is a highly orchestrated process. The involvement of the DNA damage response factors in DNA damage response depends on their biochemical reactions with each other and with chromatin. Using online live-cell imaging combined with heavy ion microbeam irradiation, we studied the response of the scaffold protein X-ray repair cross-complementary protein 1 (XRCC1) at the localized DNA damage in charged particle irradiated HT1080 cells expressing XRCC1-tagged RFP. The results showed that XRCC1 was recruited to the DNA damage with ultrafast kinetics in a poly ADP-ribose polymerase-dependent manner. The consecutive reaction model well explained the response of XRCC1 at ion hits, and we found that the XRCC1 recruitment was faster and dissociation was slower in the G2 phase than those in the G1 phase. The fractionated irradiation of the same cells resulted in accelerated dissociation at the previous damage sites, and the dissociated XRCC1 immediately recycled with a higher recruitment efficiency. Our data revealed XRCC1's new rescue mechanism and its high turnover in DNA damage response, which benefits our understanding of the biochemical mechanism in DNA damage response.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Rayos X , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
The dynamic structure of nuclear chromatin and its regulation in the formation of repair complex is essential in DNA damage response and repair. Using single molecule localization microscopy STORM this work discovered that the nuclear chromatin organization was relaxed from 200 to 400 nm thick irregular frame and remodeled to dispersed sub-100 nm structure in HeLa cells after X-ray irradiation. The DSB repair factors (γ-H2AX, MDC1, 53BP1) showed distribution as microscale-colocalized and nanoscale interlaced substructure in the DSB repair complex. The dual-color nanoscopic imaging of γ-H2AX and chromatin at the DSB sites suggest that DNA damage response and repair cascade are chromatin structure-dependent and also partly dependent on the distance to the DSB sites. The sub-100 nm structure of fibers and nanoclusters of the relaxed nuclear chromatin and the DSB repair factors highly resembled the cross-section view of chromatin organization. These data demonstrated the polymorphic and dynamic behavior of the chromatin organization in vivo, and provided nanoscale insight into the interplay between chromatin remodeling and DNA damage response and DNA repair.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN , Imagen Individual de Molécula , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Cromatina/efectos de la radiación , ADN/metabolismo , ADN/efectos de la radiación , Células HeLa , Histonas/metabolismo , Humanos , Radiación Ionizante , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Rectified ion transport in nanochannels is the basis of ion channels in biological cells and has inspired emerging nanochannel applications in ion separation, Coulter counters, and biomolecule detection and nanochannel energy harvesters. In this work we fabricated a polyethylene terephthalate (PET) conical nanochannel using latent ion track etching technique and then systematically studied the ion transport and influence of cation species on the nanochannel surface with cyclic I-V measurement. We discovered the electrical regulation of the reversible and irreversible modification of the nanochannel transportation by bivalent and trivalent cations, revealing the existence of the switching threshold voltage which can control the current rectification in bivalent solution. The proposed mechanism of the transport state transition in the PET nanochannel mimics behaviors of voltage-gated biological ion channels. These findings provide new insight into the understanding of the ion channel signaling and translocation control of charged particles in nanochannel applications.