Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Mitochondrion ; 13(4): 350-6, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23591384

RESUMEN

Homologous recombination is essential for productive DNA replication particularly under stress conditions. We previously demonstrated a stress-induced recruitment of Rad51 to mitochondria and a critical need for its activity in the maintenance of mitochondrial DNA (mtDNA) copy number. Using the human osteosarcoma cell line U20S, we show in the present study that recruitment of Rad51 to mitochondria under stress conditions requires ongoing mtDNA replication. Additionally, Rad51 levels in mitochondria increase in cells recovering from mtDNA depletion. Our findings highlight an important new role for Rad51 in supporting mtDNA replication, and further promote the idea that recombination is indispensable for sustaining DNA synthesis under conditions of replication stress.


Asunto(s)
Replicación del ADN , ADN Mitocondrial/biosíntesis , Mitocondrias/metabolismo , Recombinasa Rad51/metabolismo , Estrés Fisiológico , Línea Celular , Humanos
2.
J Cell Physiol ; 227(1): 383-9, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21503884

RESUMEN

Positive transcription elongation factor b (P-TEFb), the complex of Cyclin T1 and CDK9, activates the transcription of many viral and eukaryotic genes at the point of mRNA elongation. The activity of P-TEFb has been implicated in the differentiation of a number of cell types, including skeletal muscle. In order to promote transcription, P-TEFb hyperphosphorylates RNA Pol II, thereby increasing its processivity. Our previous work identified histone H1 as a P-TEFb substrate during HIV-1 and immediate-early transcription. Here, we examine the role of P-TEFb phosphorylation of histone H1 during differentiation, using the myoblast cell line C2C12 as a model for skeletal muscle differentiation. We found that H1 phosphorylation is elevated in differentiating C2C12, and this phosphorylation is sensitive to P-TEFb inhibition. H1 phosphorylation was also necessary for the induction of three muscle marker genes that require P-TEFb for expression. Additionally, ChIP experiments demonstrate that H1 dissociates from muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. We determine that both P-TEFb activity and H1 phosphorylation are necessary for the full differentiation of C2C12 myoblasts into myotubes.


Asunto(s)
Diferenciación Celular/genética , Histonas/genética , Músculo Esquelético/citología , Mioblastos/citología , Factor B de Elongación Transcripcional Positiva/genética , Animales , Western Blotting , Línea Celular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Histonas/metabolismo , Ratones , Mioblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Reacción en Cadena de la Polimerasa , Factor B de Elongación Transcripcional Positiva/metabolismo , Transfección
3.
Mol Microbiol ; 78(6): 1523-38, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21143322

RESUMEN

The wild-type Escherichia coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to sixfold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to fourfold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50-fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of E. coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function - filament formation and the inherent DNA pairing activity of the formed filaments.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Recombinación Genética , Secuencias de Aminoácidos , Conjugación Genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Mutación , Rec A Recombinasas/genética
4.
J Biol Chem ; 285(25): 18984-90, 2010 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-20413593

RESUMEN

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.


Asunto(s)
ADN Mitocondrial/genética , Genoma , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Ciclo Celular , Línea Celular Tumoral , Citoplasma/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Mitocondrias/metabolismo , Estrés Oxidativo , Recombinasa Rad51/metabolismo , Fracciones Subcelulares/metabolismo
5.
J Biol Chem ; 284(46): 31945-52, 2009 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-19783859

RESUMEN

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.


Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Daño del ADN , ADN de Neoplasias/genética , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Citoplasma/genética , Citoplasma/efectos de la radiación , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Genes BRCA2 , Células HCT116 , Células HeLa , Humanos , Immunoblotting , Rayos Infrarrojos , Señales de Localización Nuclear , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinasa Rad51/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares
6.
Methods ; 48(1): 63-71, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19245833

RESUMEN

Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage.


Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas/química , Proteínas/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Línea Celular , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Riñón/citología , Microscopía Fluorescente , Proteínas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transfección
7.
Biochemistry ; 46(11): 3566-75, 2007 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-17302439

RESUMEN

The human Rad51 protein requires ATP for the catalysis of DNA strand exchange, as do all Rad51 and RecA-like recombinases. However, understanding the specific mechanistic requirements for ATP binding and hydrolysis has been complicated by the fact that ATP appears to have distinctly different effects on the functional properties of human Rad51 versus yeast Rad51 and bacterial RecA. Here we use RNAi methods to test the function of two ATP binding site mutants, K133R and K133A, in human cells. Unexpectedly, we find that the K133A mutant is functional for repair of DNA double-strand breaks when endogenous Rad51 is depleted. We also find that the K133A protein maintains wild-type-like DNA binding activity and interactions with Brca2 and Xrcc3, properties that undoubtedly promote its DNA repair capability in the cell-based assay used here. Although a Lys to Ala substitution in the Walker A motif is commonly assumed to prevent ATP binding, we show that the K133A protein binds ATP, but with an affinity approximately 100-fold lower than that of wild-type Rad51. Our data suggest that ATP binding and release without hydrolysis by the K133A protein act as a mechanistic surrogate in a catalytic process that applies to all RecA-like recombinases. ATP binding promotes assembly and stabilization of a catalytically active nucleoprotein filament, while ATP hydrolysis promotes filament disassembly and release from DNA.


Asunto(s)
Reparación del ADN/fisiología , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Línea Celular , Humanos , Interferencia de ARN
8.
Biochemistry ; 45(45): 13537-42, 2006 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-17087507

RESUMEN

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.


Asunto(s)
Nucleoproteínas/metabolismo , Rec A Recombinasas/fisiología , Adenosina Trifosfatasas/metabolismo , Dimerización , Nucleoproteínas/ultraestructura , Rec A Recombinasas/genética , Proteínas Recombinantes de Fusión/metabolismo
9.
Proc Natl Acad Sci U S A ; 103(48): 18137-42, 2006 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-17110439

RESUMEN

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form gamma-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that gamma-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of gamma-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of gamma-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.


Asunto(s)
Cromatina/química , Cromatina/metabolismo , Daño del ADN , Histonas/metabolismo , Microscopía/métodos , Fosfatos/análisis , Núcleo Celular , Cromatina/genética , Células HeLa , Humanos , Familia de Multigenes
10.
J Cell Biochem ; 96(6): 1095-109, 2005 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-16215984

RESUMEN

Rad51-catalyzed homologous recombination is an important pathway for repair of DNA double strand breaks and maintenance of genome integrity in vertebrate cells. Five proteins referred to as Rad51 paralogs promote Rad51 activity and are proposed to act at various, and in some cases, multiple stages in the recombination pathway. Imaging studies of native Rad51 have revealed its cellular response to DNA damage, yet visualization of the paralog proteins has met with limited success. In this study, we are able to detect endogenous Rad51C and Xrcc3 in human cells. In an effort to determine how Rad51, Rad51C, and Xrcc3 influence the pattern of localization of each other over the time course of DNA damage and repair, we have made the unexpected observation that Rad51 degradation via the ubiquitin-mediated proteasome pathway occurs as a natural part of recombinational DNA repair. Additionally, we find that Rad51C plays an important role in regulating this process. This article contains supplementary material, which may be viewed at the Journal of Cellular Biochemistry website at http://www.interscience.wiley.com/jpages/0730-2312/suppmat/index.html.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Ubiquitina/metabolismo , Animales , Células Cultivadas , Daño del ADN/fisiología , Daño del ADN/efectos de la radiación , Reparación del ADN/fisiología , Regulación de la Expresión Génica , Células HeLa , Humanos , Microscopía Confocal , ARN Interferente Pequeño/metabolismo , Ubiquitina/farmacología
11.
J Mol Biol ; 345(2): 239-49, 2005 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-15571718

RESUMEN

Human Rad52 (HsRad52) is a DNA-binding protein (418 residues) that promotes the catalysis of DNA double strand break repair by the Rad51 recombinase. HsRad52 self-associates to form ring-shaped oligomers as well as higher order complexes of these rings. Analysis of the structural and functional organization of protein domains suggests that many of the determinants of DNA binding lie within the N-terminal 85 residues. Crystal structures of two truncation mutants, HsRad52(1-212) and HsRad52(1-209) support the idea that this region makes up an important part of the DNA binding domain. Here, we report the results of saturating alanine scanning mutagenesis of the N-terminal domain of full-length HsRad52 in which we identify residues that are likely involved in direct contact with single-stranded DNA (ssDNA). Our results largely agree with the position of side-chains seen in the crystal structures but also suggest that certain DNA binding and cross-subunit interactions differ between the 11 subunit ring in the crystal structures of the truncation mutant proteins versus the seven subunit ring formed by full-length HsRad52.


Asunto(s)
ADN , Proteínas Serina-Treonina Quinasas/química , Alanina/química , Secuencia de Aminoácidos , Arginina/química , Catálisis , Línea Celular Transformada , Quinasa de Punto de Control 2 , Cromatografía en Gel , Cristalografía por Rayos X , ADN/química , Daño del ADN , Reparación del ADN , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación , Plásmidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Recombinasa Rad51 , Proteína Recombinante y Reparadora de ADN Rad52 , Recombinación Genética , Homología de Secuencia de Aminoácido
12.
J Cell Biochem ; 93(3): 429-36, 2004 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-15372620

RESUMEN

Rad51-mediated homologous recombination (HR) is essential for maintenance of genome integrity. The Xrcc3 protein functions in HR DNA repair, and studies suggest it has multiple roles at different stages in this pathway. Defects in vertebrate XRCC3 result in elevated levels of spontaneous and DNA damage-induced chromosomal abnormalities, as well as increased sensitivity to DNA damaging agents. Formation of DNA damaged-induced nuclear Rad51 foci requires Xrcc3 and the other Rad51 paralog proteins (Rad51B, Rad51C, Rad51D, Xrcc2), thus supporting a model in which an early function of Xrcc3 involves promoting assembly of active Rad51 repair complexes. However, it is not known whether Xrcc3 or other Rad51 paralog proteins accumulate at DNA breaks, and if they do whether their stable association with breaks requires Rad51. Here we report for the first time that Xrcc3 forms distinct foci in human cells and that nuclear Xrcc3 begins to localize at sites of DNA damage within 10 min after radiation treatment. RNAi-mediated knock down of Rad51 has no effect on the DNA damage-induced localization of Xrcc3 to DNA breaks. Our data are consistent with a model in which Xrcc3 associates directly with DNA breaks independent of Rad51, and subsequently facilitates formation of the Rad51 nucleoprotein filament.


Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Rayos gamma/efectos adversos , Humanos , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51
13.
Crit Rev Biochem Mol Biol ; 38(5): 385-432, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-14693725

RESUMEN

The bacterial RecA protein participates in a remarkably diverse set of functions, all of which are involved in the maintenance of genomic integrity. RecA is a central component in both the catalysis of recombinational DNA repair and the regulation of the cellular SOS response. Despite the mechanistic differences of its functions, all require formation of an active RecA/ATP/DNA complex. RecA is a classic allosterically regulated enzyme, and ATP binding results in a dramatic increase in DNA binding affinity and a cooperative assembly of RecA subunits to form an ordered, helical nucleoprotein filament. The molecular events that underlie this ATP-induced structural transition are becoming increasingly clear. This review focuses on descriptions of our current understanding of the molecular design and allosteric regulation of RecA. We present a comprehensive list of all published recA mutants and use the results of various genetic and biochemical studies, together with available structural information, to develop ideas regarding the design of RecA functional domains and their catalytic organization.


Asunto(s)
Bacterias/genética , Reparación del ADN , Rec A Recombinasas/química , Rec A Recombinasas/genética , Regulación Alostérica , Sitios de Unión , Secuencia de Consenso , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli/fisiología , Mutación , Rec A Recombinasas/metabolismo , Recombinación Genética , Respuesta SOS en Genética
14.
J Biol Chem ; 277(48): 46172-8, 2002 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-12226092

RESUMEN

The human Rad52 protein self-associates to form ring-shaped oligomers, as well as higher order complexes of these rings. We have shown previously that there are two experimentally separable self-association domains in HsRad52, one in the N terminus (residues 1-192) responsible for assembly of individual subunits into rings, and one in the C terminus (residues 218-418) responsible for higher order oligomerization of rings. Earlier studies suggest that the higher order complexes promote DNA end-joining, and others suggest that these complexes are relevant to in vivo Rad52 function. In this study we demonstrate that although inherent binding to single-stranded DNA depends on neither higher order complexes of Rad52 rings nor the ring-shaped oligomers themselves, higher order complexes are important for activities involving simultaneous interaction with more than one DNA molecule. This provides biochemical support for what may be an important in vivo function of Rad52.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencia de Bases , Biopolímeros , Cartilla de ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Humanos , Microscopía Electrónica
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...