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1.
Nucleic Acids Res ; 45(14): 8493-8507, 2017 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-28591846

RESUMEN

We demonstrate an application of atomic force microscopy (AFM) for the structural analysis of long single-stranded RNA (>1 kb), focusing on 28S ribosomal RNA (rRNA). Generally, optimization of the conditions required to obtain three-dimensional (3D) structures of long RNA molecules is a challenging or nearly impossible process. In this study, we overcome these limitations by developing a method using AFM imaging combined with automated, MATLAB-based image analysis algorithms for extracting information about the domain organization of single RNA molecules. We examined the 5 kb human 28S rRNA since it is the largest RNA molecule for which a 3D structure is available. As a proof of concept, we determined a domain structure that is in accordance with previously described secondary structural models. Importantly, we identified four additional small (200-300 nt), previously unreported domains present in these molecules. Moreover, the single-molecule nature of our method enabled us to report on the relative conformational variability of each domain structure identified, and inter-domain associations within subsets of molecules leading to molecular compaction, which may shed light on the process of how these molecules fold into the final tertiary structure.


Asunto(s)
Imagenología Tridimensional/métodos , Microscopía de Fuerza Atómica/métodos , Conformación de Ácido Nucleico , ARN Ribosómico 28S/química , Algoritmos , Sitios de Unión/genética , Células HeLa , Humanos , Cinética , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , Reproducibilidad de los Resultados
2.
J Biol Chem ; 288(31): 22437-50, 2013 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-23779106

RESUMEN

Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.


Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de la Membrana/metabolismo , Rec A Recombinasas/metabolismo , Bacillus subtilis/genética , Biocatálisis , Cromosomas Bacterianos , Unión Proteica , Recombinación Genética
3.
Cytogenet Genome Res ; 144(4): 255-263, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25766002

RESUMEN

Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.


Asunto(s)
Síndrome de Bloom/genética , Inestabilidad Cromosómica , RecQ Helicasas/metabolismo , Intercambio de Cromátides Hermanas , Síndrome de Bloom/metabolismo , Células Cultivadas , Cromosomas Humanos/genética , Humanos , Método de Montecarlo , Mutación , Fosforilación , RecQ Helicasas/genética
4.
Cell Biol Int ; 34(9): 893-9, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20507282

RESUMEN

The FA (Fanconi anaemia) FANCD2 protein is pivotal in the cellular response to DNA interstrand cross-links. Establishing cells expressing exogenous FANCD2 has proven to be difficult compared with other DNA repair genes. We find that in transformed normal human fibroblasts, exogenous nuclear expression of FANCD2 induces apoptosis, dependent specifically on exons 10-13. This is the same region required for interaction with the histone acetyltransferase, Tip60. Deletion of exons 10-13 from FANCD2 N-terminal constructs (nucleotides 1-1100) eliminates the binary interaction with Tip60 and the cellular apoptotic response; moreover, cells can stably express FANCD2 at high levels if Tip60 is depleted. The results indicate that FANCD2-sponsored apoptosis requires an interaction with Tip60 and depends on Tip60.


Asunto(s)
Apoptosis , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Histona Acetiltransferasas/metabolismo , Núcleo Celular/metabolismo , Células Clonales , Ensayo de Unidades Formadoras de Colonias , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Fluorescencia , Células HeLa , Humanos , Lisina Acetiltransferasa 5 , Señales de Localización Nuclear/metabolismo , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Relación Estructura-Actividad
5.
Nucleic Acids Res ; 35(18): 6115-23, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17804464

RESUMEN

The human SNM1 protein is a member of a highly conserved group of proteins catalyzing the hydrolysis of nucleic acid substrates. Although overproduction is unstable in mammalian cells, we have overproduced a recombinant hSNM1 protein in an insect cell system. The protein is a single-strand 5'-exonuclease, like its yeast homolog. The enzyme utilizes either DNA or RNA substrates, requires a 5'-phosphate moiety, shows very little activity on double-strand substrates, and functions at a size consistent with a monomer. The exonuclease activity requires the conserved beta-lactamase domain; site-directed mutagenesis of a conserved aspartate inactivates the exonuclease.


Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Ácido Aspártico/genética , Línea Celular , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , ADN de Cadena Simple/metabolismo , Drosophila melanogaster/citología , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Fibroblastos/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfatos/metabolismo
6.
J Biochem ; 165(6): 465-470, 2019 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-30821334

RESUMEN

Fanconi anemia (FA) is a devastating hereditary disorder with impaired genome stability resulting in physical abnormalities, gradual loss of hematopoietic stem cells and development of tumours and leukaemia. It has been suggested that functions of FA genes are required to maintain genome stability by counteracting endogenous metabolites, such as aldehydes, that damage DNA and stall replication forks. Recent studies have implicated co-transcriptional R-loops, consisting of a DNA:RNA hybrid and displaced single-stranded DNA, as one of the potential endogenous sources that induce genome instability and the FA phenotype. This review focuses on recent literature, including our own, regarding the interplay between FA proteins and R-loops, and will provide readers with a concise summary of this rapidly evolving field.


Asunto(s)
Anemia de Fanconi/genética , Inestabilidad Genómica , Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Humanos
7.
DNA Repair (Amst) ; 4(2): 163-70, 2005 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-15590324

RESUMEN

Interstrand cross-links (ICL) in DNA arise from bifunctional alkylating agents, including nitrogen mustards, mitomycin C and psoralens. Such adducts prevent normal transcription or replication and are mutagenic. Therefore, cellular mechanisms for removing ICL damage are needed to maintain genome stability. Normal ICL repair requires the action of a number of genes, some specific for such damage. The yeast Snm1 protein is one such protein, but its function has been unknown. Incision for ICL repair is normal in mutants lacking Snm1, so it appears to act after the earliest steps. We have used recombinant SNM1 constructs in an Escherichia coli (E. coli) expression system to demonstrate that the yeast gene encodes a 5'-exonuclease. The exonuclease activity is required for Snm1 to be functional in ICL repair.


Asunto(s)
Reparación del ADN , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Fosfodiesterasa I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/aislamiento & purificación , Endodesoxirribonucleasas , Escherichia coli/enzimología , Expresión Génica , Proteínas Nucleares/aislamiento & purificación , Fosfodiesterasa I/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación
8.
DNA Repair (Amst) ; 12(3): 162-76, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23380520

RESUMEN

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3'-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes.


Asunto(s)
Bacillus subtilis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Bacteriano/genética , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/fisiología , Cromosomas Bacterianos/genética , Reparación del ADN , Enzimas de Restricción del ADN/fisiología , ADN Bacteriano/metabolismo , ADN Bacteriano/ultraestructura , Fosforilación , Procesamiento Proteico-Postraduccional , Reparación del ADN por Recombinación
9.
Int J Cell Biol ; 2012: 494902, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22229032

RESUMEN

Various nuclear functional complexes contain cytoskeletal proteins as regulatory subunits; for example, nuclear actin participates in transcriptional complexes, and actin-related proteins are integral to chromatin remodeling complexes. Nuclear complexes such as these are involved in both basal and adaptive nuclear functions. In addition to nuclear import via classical nuclear transport pathways or passive diffusion, some large cytoskeletal proteins spontaneously migrate into the nucleus in a karyopherin-independent manner. The balance of nucleocytoplasmic distribution of such proteins can be altered by several factors, such as import versus export, or capture and release by complexes. The resulting accumulation or depletion of the nuclear populations thereby enhances or attenuates their nuclear functions. We propose that such molecular dynamics constitute a form of cytoskeleton-modulated regulation of nuclear functions which is mediated by the translocation of cytoskeletal components in and out of the nucleus.

10.
J Biol Chem ; 283(15): 9844-51, 2008 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-18263878

RESUMEN

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.


Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Reparación del ADN/fisiología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Histona Acetiltransferasas/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Línea Celular , Núcleo Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Histona Acetiltransferasas/genética , Humanos , Lisina Acetiltransferasa 5 , Mitomicina/farmacología , Ubiquitinación/efectos de los fármacos , Ubiquitinación/fisiología
11.
Hum Mol Genet ; 11(21): 2591-7, 2002 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-12354784

RESUMEN

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.


Asunto(s)
Proteína BRCA1/metabolismo , Proteínas de Unión al ADN , Proteínas/metabolismo , Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Humanos , Técnicas In Vitro , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Técnicas del Sistema de Dos Híbridos
12.
Hum Mol Genet ; 13(12): 1241-8, 2004 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-15115758

RESUMEN

Fanconi anaemia (FA) is a chromosomal instability disorder characterized by cellular sensitivity to DNA interstrand crosslinking agents and a high risk of cancer. Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the monoubiquitination of the FANCD2 protein. FANCD2 complexes and colocalizes with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined. We used yeast two-hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair. FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1. However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9. FANCD2 and BRCA2 can be coimmunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo. Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wild-type BRCA2 function. FANCD2 colocalized with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and colocalized very tightly with PCNA after treatment with hydroxyurea. These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion.


Asunto(s)
Proteína BRCA2/metabolismo , Daño del ADN , Proteínas Nucleares/metabolismo , Animales , Proteína BRCA2/genética , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cricetinae , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Humanos , Inmunoprecipitación , Proteínas Nucleares/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Recombinasa Rad51 , Técnicas del Sistema de Dos Híbridos , Levaduras
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