RESUMO
Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação/efeitos da radiação , Sequência de Bases , Montagem e Desmontagem da Cromatina , DNA Fúngico/química , Proteínas de Ligação a DNA/química , Histonas/química , Lisina/química , Modelos Moleculares , Nucleossomos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Raios UltravioletaRESUMO
Histone octamers are the basic building blocks of chromatin and are platforms for diverse genetic mechanisms. We report a simple method for preparing recombinant histone octamers by overexpressing all four histones from a single polycistronic vector followed by standard chromatography under native conditions. This approach reduces the time needed for the octamer preparation to a single day and should be applicable to making a variety of unmodified and modified histone octamers.
Assuntos
Proteínas de Bactérias/genética , Cromatina/genética , Vetores Genéticos/genética , Histonas/genética , Bactérias , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatina/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Vetores Genéticos/química , Histonas/isolamento & purificação , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased "intrinsic exposure" of nucleosome-associated DNA lesions in chromatin to repair proteins.
Assuntos
Dano ao DNA , Reparo do DNA , Nucleossomos/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/metabolismo , Nucleossomos/efeitos da radiação , Xenopus laevisRESUMO
Chondroitin sulfate (CS) is a kind of linear polysaccharide that is covalently linked to proteins to form proteoglycans. Chondroitin sulfate proteoglycans (CSPGs) consist of a core protein, with one or more CS chains covalently attached. CSPGs are precisely regulated and they exert a variety of physiological functions by binding to adhesion molecules and growth factors. Widely distributed in the nervous system in human body, CSPGs contribute to the major component of extracellular matrix (ECM), where they play an important role in the development and maturation of the nervous system, as well as in the pathophysiological response to damage to the central nervous system (CNS). While there are more than 30 types of CSPGs, this review covers the roles of the most important ones, including versican, aggrecan, neurocan and NG2 in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. The updated reports of the treatment of neurodegenerative diseases are involving CSPGs.
Assuntos
Proteoglicanas de Sulfatos de Condroitina , Doenças Neurodegenerativas , Sistema Nervoso Central , Matriz Extracelular , HumanosRESUMO
WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA/química , Modelos Moleculares , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Núcleo Celular/química , Sequência Conservada , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Ligação de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Ácido Salicílico/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Zinco/químicaRESUMO
The C-terminal WRKY domain of Arabidopsis thaliana WRKY1 protein, a transcription factor, was cloned and expressed. The expressed protein was then purified and crystallized. The preliminary X-ray analysis was undertaken. The crystal diffracted to 2.50 A resolution in-house and belongs to space group P2(1) with unit-cell parameters a=64.10 A, b=34.88 A, c=114.72 A, beta=90.49 degrees .