RESUMEN

Birt-Hogg-Dubé (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase their tumorigenic potential, forming larger spheroids in non-adherent conditions. Transcriptomic and proteomic analysis uncovered links between FLCN, cell cycle control and the DNA damage response (DDR) machinery. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen uncovered FLCN binding to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked elevation of γH2AX and RB1 phosphorylation. Both CCND1 and RB1 phosphorylation remained raised during DNA damage, showing an association with defective cell cycle control with FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest by DNA damage. This work implicates that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

RESUMEN

Glioblastoma (GBM) is an aggressive and devastating primary brain cancer which responds very poorly to treatment. The average survival time of patients is only 14-15 months from diagnosis so there is a clear and unmet need for the development of novel targeted therapies to improve patient outcomes. The multifunctional cytokine TGFß plays fundamental roles in development, adult tissue homeostasis, tissue wound repair and immune responses. Dysfunction of TGFß signalling has been implicated in both the development and progression of many tumour types including GBM, thereby potentially providing an actionable target for its treatment. This review will examine TGFß signalling mechanisms and their role in the development and progression of GBM. The targeting of TGFß signalling using a variety of approaches including the TGFß binding protein Decorin will be highlighted as attractive therapeutic strategies.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Decorina/metabolismo , Glioblastoma/tratamiento farmacológico , Humanos , Microambiente Tumoral

RESUMEN

Frank-Ter Haar syndrome (FTHS), Winchester syndrome (WS), and multicentric osteolysis, nodulosis, and arthropathy (MONA) are ultra-rare multisystem disorders characterized by craniofacial malformations, reduced bone density, skeletal and cardiac anomalies, and dermal fibrosis. These autosomal recessive syndromes are caused by homozygous mutation or deletion of respectively SH3PXD2B (SH3 and PX Domains 2B), MMP14 (matrix metalloproteinase 14), or MMP2. Here, we give an overview of the clinical features of 63 previously reported patients with an SH3PXD2B, MMP14, or MMP2 mutation, demonstrating considerable clinical overlap between FTHS, WS, and MONA. Interestingly, the protein products of SH3PXD2B, MMP14, and MMP2 directly cooperate in collagen remodeling. We review animal models for these three disorders that accurately reflect the major clinical features and likewise show significant phenotypical similarity with each other. Furthermore, they demonstrate that defective collagen remodeling is central in the underlying pathology. As such, we propose a nosological revision, placing these SH3PXD2B, MMP14, and MMP2 related syndromes in a novel "defective collagen-remodelling spectrum (DECORS)". In our opinion, this revised nosology better reflects the central role for impaired collagen remodeling, a potential target for pharmaceutical intervention.

Asunto(s)

Colágeno/genética , Síndrome de Hajdu-Cheney/diagnóstico , Síndrome de Hajdu-Cheney/genética , Mutación , Fenotipo , Alelos , Animales , Colágeno/química , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo

RESUMEN

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.

Asunto(s)

Conexinas/genética , Eritroqueratodermia Variable/genética , Mutación Missense , Muerte Celular , Membrana Celular/efectos de los fármacos , Células Cultivadas , Conexina 43/biosíntesis , Conexina 43/genética , Retículo Endoplásmico/ultraestructura , Epidermis/patología , Eritroqueratodermia Variable/patología , Genes Dominantes , Estudios de Asociación Genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacología , Células HeLa , Humanos , Queratinocitos , Necrosis , Transporte de Proteínas

RESUMEN

Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14's transport to the cell membrane. Instead, it partially impairs MMP14's proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14's catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density owing to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.

Asunto(s)

Anomalías Múltiples/genética , Contractura/genética , Opacidad de la Córnea/genética , Anomalías Craneofaciales/genética , Trastornos del Crecimiento/genética , Metaloproteinasa 14 de la Matriz/genética , Osteólisis/genética , Osteoporosis/genética , Anomalías Múltiples/fisiopatología , Alelos , Animales , Dominio Catalítico/genética , Contractura/fisiopatología , Opacidad de la Córnea/fisiopatología , Anomalías Craneofaciales/fisiopatología , Técnicas de Inactivación de Genes , Trastornos del Crecimiento/fisiopatología , Humanos , Ratones , Osteólisis/fisiopatología , Osteoporosis/fisiopatología , Fenotipo , Pez Cebra

RESUMEN

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

Asunto(s)

Proteínas Quinasas Activadas por AMP/genética , Glucógeno/metabolismo , Osmorregulación/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Glucógeno/genética , Glucógeno Fosforilasa/genética , Glucógeno Sintasa/genética , Humanos , Ratones , Mutación , Concentración Osmolar

RESUMEN

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Homólogo de la Proteína 1 Relacionada con la Autofagia , Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patología , Proteínas Portadoras/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/deficiencia , Proteína Sequestosoma-1 , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/deficiencia

RESUMEN

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Síndrome de Birt-Hogg-Dubé/etiología , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Línea Celular , Transformación Celular Neoplásica , Metabolismo Energético , Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética

RESUMEN

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Asunto(s)

Síndrome de Birt-Hogg-Dubé/fisiopatología , Cilios/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Síndrome de Birt-Hogg-Dubé/genética , Línea Celular , Polaridad Celular , Proliferación Celular , Centrosoma/fisiología , Cilios/patología , Humanos , Riñón/fisiología , Microtúbulos/fisiología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Vía de Señalización Wnt

RESUMEN

Birt-Hogg-Dubé syndrome (MIM #135150) is characterized by the development of benign skin tumours called fibrofolliculomas, pulmonary cysts that may lead to pneumothorax and a high risk of developing kidney cancer. BHD is caused by mutations affecting the highly conserved protein folliculin (FLCN), which probably has a role in intracellular transport. Most of the research effort directed towards BHD has focused on understanding how loss of FLCN causes kidney cancer. The cutaneous manifestations have received comparatively little attention. Although understandable, it is unfortunate, as the fibrofolliculomas are highly accessible and thus potentially are an excellent system for trying to understand the basic pathobiology of BHD. Also, patients can be very much burdened by the cosmetic consequences of having hundreds of facial skin tumours. Our lack of insight into what drives fibrofolliculoma growth translates into a very limited therapeutic arsenal. Thus, paying attention to fibrofolliculomas has both basic science and practical benefits. In this review, we will discuss the state of the art regarding our understanding of fibrofolliculoma pathogenesis and indicate future directions for research.

Asunto(s)

Síndrome de Birt-Hogg-Dubé/complicaciones , Enfermedades de la Piel/etiología , Humanos , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/terapia

RESUMEN

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition due to germline FLCN (folliculin) mutations, characterized by skin fibrofolliculomas, lung cysts, pneumothorax and renal cancer. We identified a de novo FLCN mutation, c.499C>T (p.Gln167X), in a patient who presented with spontaneous pneumothorax. Subsequently, typical skin features and asymptomatic renal cancer were diagnosed. Probably, de novo FLCN mutations are rare. However, they may be under-diagnosed if BHD is not considered in sporadic patients who present with one or more of the syndromic features. Genetic and immunohistochemical analysis of the renal tumour indicated features compatible with a tumour suppressor role of FLCN. The finding that mutant FLCN was expressed in the tumour might indicate residual functionality of mutant FLCN, a notion which will be explored in future studies.

Asunto(s)

Mutación de Línea Germinal/genética , Neoplasias Renales/genética , Neumotórax/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética , Adulto , Humanos , Técnicas para Inmunoenzimas , Neoplasias Renales/diagnóstico , Imagen por Resonancia Magnética , Masculino , Neumotórax/diagnóstico , Pronóstico , Tomografía Computarizada por Rayos X

RESUMEN

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Asunto(s)

Daño del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Ubiquitina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Biología Computacional , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Xerodermia Pigmentosa/genética , Dedos de Zinc , ADN Polimerasa iota

RESUMEN

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.

Asunto(s)

Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Cafeína/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Daño del ADN , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Modelos Genéticos , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta , Xerodermia Pigmentosa , ADN Polimerasa iota

RESUMEN

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.

Asunto(s)

Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Transporte Activo de Núcleo Celular , Animales , Cafeína/toxicidad , Línea Celular , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Núcleo Celular/enzimología , ADN/efectos de los fármacos , ADN/efectos de la radiación , Daño del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Genes Reporteros , Prueba de Complementación Genética , Humanos , Microscopía Fluorescente , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Spodoptera/citología , Transfección , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/patología , ADN Polimerasa iota