RESUMEN
DNA interstrand cross-links (ICLs) represent a physical barrier to the progression of cellular machinery involved in DNA metabolism. Thus, this type of adduct represents a serious threat to genomic stability and as such, several DNA repair pathways have evolved in both higher and lower eukaryotes to identify this type of damage and restore the integrity of the genetic material. Human cells possess a specialized ICL-repair system, the Fanconi anemia (FA) pathway. Conversely yeasts rely on the concerted action of several DNA repair systems. Recent work in higher eukaryotes identified and characterized a novel conserved FA component, FAN1 (Fanconi anemia-associated nuclease 1, or FANCD2/FANCI-associated nuclease 1). In this study, we characterize Fan1 in the yeast Schizosaccharomyces pombe. Using standard genetics, we demonstrate that Fan1 is a key component of a previously unidentified ICL-resolution pathway. Using high-throughput synthetic genetic arrays, we also demonstrate the existence of a third pathway of ICL repair, dependent on the SUMO E3 ligase Pli1. Finally, using sequence-threaded homology models, we predict and validate key residues essential for Fan1 activity in ICL repair.
Asunto(s)
Reparación del ADN , Esterasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Ubiquitina-Proteína Ligasas/metabolismo , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Esterasas/química , Esterasas/genética , Evolución Molecular , Ensayos Analíticos de Alto Rendimiento , Ligasas , Modelos Moleculares , Tasa de Mutación , Estructura Terciaria de Proteína , Recombinación Genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF. METHODS: Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation. RESULTS: Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16. CONCLUSION: These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Silenciador del Gen , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Unión Proteica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.
Asunto(s)
Amidas/farmacología , Comunicación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Piridinas/farmacología , Amidas/síntesis química , Amidas/química , Animales , Aurora Quinasa A , Aurora Quinasas , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Clonación Molecular/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Células 3T3 NIH , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT(5)G(4)CT(4)C) and the RNA equivalent r(GU(5)G(4)CU(4)C), the latter interaction being important for translational repression. We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein. Binding of gene 5 protein to the tetraplex leads to formation of a approximately 170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120-130 A. A model of the complex is presented that is consistent with the data obtained. It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression.
Asunto(s)
ADN/metabolismo , Inovirus/química , Inovirus/metabolismo , Proteínas Virales/metabolismo , Cromatografía en Gel , Dicroismo Circular , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/virología , G-Cuádruplex , Regulación Viral de la Expresión Génica , Guanina/química , Luz , Modelos Biológicos , Oligonucleótidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Dispersión de Radiación , Ultracentrifugación , Rayos Ultravioleta , Rayos XRESUMEN
The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.