RESUMEN
Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required.
Asunto(s)
Cromatina/metabolismo , Daño del ADN/fisiología , Reparación del ADN/fisiología , Genes cdc/fisiología , Inestabilidad Genómica/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Animales , Histonas/metabolismo , Humanos , FosforilaciónRESUMEN
Fluorescent protein labelling, as well as impressive progress in live cell imaging have revolutionised the view on how essential nuclear functions like gene transcription regulation and DNA repair are organised. Here, we address questions like how DNA-interacting molecules find and bind their target sequences in the vast amount of DNA. In addition, we discuss methods that have been developed for quantitative analysis of data from fluorescence recovery after photobleaching experiments (FRAP).
Asunto(s)
Sitios de Unión , Cromatina/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Proteínas Nucleares/metabolismo , Reparación del ADN , Difusión , Estudios de Evaluación como Asunto , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Unión ProteicaRESUMEN
Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) eta and iota are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that poleta is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of poleta in foci, it results in an increased residence time in foci. poliota is even more mobile than poleta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both poleta and poliota diffuse through the cell but that they are transiently immobilized for approximately 150 ms, with a larger proportion of poleta than poliota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of poleta but not poliota. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.
Asunto(s)
Cromatina/química , ADN Polimerasa Dirigida por ADN/metabolismo , Conformación de Ácido Nucleico , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Antraquinonas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/metabolismo , ADN/química , ADN/metabolismo , ADN/efectos de la radiación , Daño del ADN , ADN Polimerasa Dirigida por ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitinación , ADN Polimerasa iotaRESUMEN
The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción/metabolismo , Animales , Células CHO , Línea Celular Transformada , Supervivencia Celular/efectos de la radiación , Transformación Celular Viral , Cricetinae , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Indoles , Cinética , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , Rayos UltravioletaRESUMEN
The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.