RESUMO
Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair.
Assuntos
Fator Estimulador de Clivagem/fisiologia , Reparo do DNA , Poliadenilação , Dano ao DNA , Células HeLa , Humanos , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Raios UltravioletaRESUMO
Cytochrome c is one of the most studied proteins probably due to its electron-transfer properties in aerobic and anaerobic respiration. Particularly, cytochrome c from bovine heart is a small protein, M(r) 12,230 Da, globular (hydrodynamic diameter of 3.4 nm), soluble in different buffer solutions, and commercially available. Despite being a quite well-studied protein and relatively easy to manipulate from the biochemical and electrochemical viewpoint, its 3D structure has never been published. In this work, the purification, crystallization and 3D structure of one of the cytochrome c isoforms is presented to 1.5 A resolution. It is also shown how the presence of isoforms made both the purification and crystallization steps difficult. Finally, a new approach for protein electrocrystallization and design of biosensors is presented.