RESUMEN
BACKGROUND: Monoclonal gammopathies (MGs) are plasma cell disorders defined by the clonal expansion of plasma cells, resulting in the characteristic excretion of a monoclonal immunoglobulin (M-protein). M-protein detection and quantification are integral parts of the diagnosis and monitoring of MGs. Novel treatment modalities impose new challenges on the traditional electrophoretic and immunochemical methods that are routinely used for M-protein diagnostics, such as interferences from therapeutic monoclonal antibodies and the need for increased analytical sensitivity to measure minimal residual disease. CONTENT: Mass spectrometry (MS) is ideally suited to accurate mass measurements or targeted measurement of unique clonotypic peptide fragments. Based on these features, MS-based methods allow for the analytically sensitive measurement of the patient-specific M-protein. SUMMARY: This review provides a comprehensive overview of the MS methods that have been developed recently to detect, characterize, and quantify M-proteins. The advantages and disadvantages of using these techniques in clinical practice and the impact they will have on the management of patients with MGs are discussed.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/sangre , Espectrometría de Masas/métodos , Paraproteinemias/diagnóstico , Anticuerpos Monoclonales/química , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión , Humanos , Paraproteinemias/patología , Péptidos/químicaRESUMEN
Estrogen-stimulated growth of the malignant human endometrium can be balanced by the differentiating properties of progesterone. To study the molecular basis behind this, gene expression profiling was performed using complementary DNA microarray analysis. In this study, the human endometrial cancer cell lines ECC-1 and PRAB-36 were used as models. The ECC-1 cell line, which expresses high levels of estrogen receptor alpha and is stimulated in growth by estrogens, was used to study estrogen regulation of gene expression. The Ishikawa sub-cell line PRAB-36, expressing both PRA and PRB, progesterone receptor isoforms, and inhibited in growth by progestagens, was used to study progesterone regulation of gene expression. Using these two well-differentiated human endometrial cancer cell lines, 148 estrogen- and 148 progesterone-regulated genes were identified. After functional classification, the estrogen- and progesterone-regulated genes could be categorized in different biologically relevant groups. Within the group of "cell growth and/or maintenance," 81 genes were clustered, from which a number of genes could be involved in arranging the cross talk that exists between estrogen and progesterone signaling. On the basis of analysis of the current findings, it is hypothesized that cross talk between estrogen and progestagen signaling does not occur by counterregulation of single genes, but rather at the level of differential regulation of different genes within the same functional families.
Asunto(s)
Neoplasias Endometriales/patología , Estrógenos/farmacología , Progesterona/farmacología , Receptores de Progesterona/metabolismo , Western Blotting , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , Estrógenos/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Progesterona/metabolismo , Receptores de Progesterona/genética , Sensibilidad y EspecificidadRESUMEN
Tibolone is a synthetic steroid with estrogenic effects on brain, vagina, and bone without stimulating the endometrium. During tibolone treatment, it is thought that the progestagenic properties of tibolone stimulate cell differentiation, which effectively counterbalances the growth-stimulating effects of the estrogenic properties of tibolone. The objective of this study was to characterize the expression profile that reflects the endometrial responses to the separated estrogenic (growth-inducing) and progestagenic (growth-inhibiting) actions of tibolone, thus gaining insight into the counteracting effect of these properties of tibolone on the endometrium. The estrogenic action of tibolone was studied in the estrogen-responsive ECC1 cell line (expressing estrogen receptor alpha), and the progestagenic action was studied in the progesterone-responsive cell line Ishikawa PRAB-36 (expressing PRA and PRB). The data showed that the progestagenic and estrogenic effects of tibolone produce different expression profiles with a narrow overlap in genes; however, both properties modulate the same biological processes. The final genetic network analysis indicated that the estrogenic effect of tibolone is potentially counterbalanced by the progestagenic metabolite of tibolone via differential regulation of similar cellular processes. For example, both progestagenic and estrogenic properties stimulate proliferation, but they exert the opposite effect on apoptosis. The apoptosis network was stimulated by the progestagenic properties of tibolone; in contrast, the estrogenic effect of tibolone suppressed the apoptosis network. The current results indicate that this differential regulation is realized through modulation of a different group of genes and rarely via contraregulation of the same set of genes.
Asunto(s)
Moduladores de los Receptores de Estrógeno/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Norpregnenos/farmacología , Progestinas/antagonistas & inhibidores , Adenocarcinoma/genética , Adenocarcinoma/patología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Estradiol/farmacología , Femenino , Perfilación de la Expresión Génica , Humanos , Red Nerviosa , Transcripción Genética/efectos de los fármacosRESUMEN
The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity. The DNA-binding specificity and the functional interactions of MlRuvA with E. coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions. MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA. Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency. These results demonstrate that the RuvA homologue of M. leprae is a functional branch-migration subunit. Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex. The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation. Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes. These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome".
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Mycobacterium leprae/enzimología , Recombinación Genética , Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Calcio/farmacología , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , ADN/química , ADN/genética , ADN/metabolismo , ADN Helicasas/metabolismo , Escherichia coli/genética , Sustancias Macromoleculares , Magnesio/metabolismo , Magnesio/farmacología , Mycobacterium leprae/genética , Unión Proteica/efectos de los fármacos , Recombinación Genética/genética , Especificidad por SustratoRESUMEN
Cancer-causing mutations often arise from gross chromosomal rearrangements (GCRs) such as translocations, which involve genetic exchange between nonhomologous chromosomes. Here we show that murine Brca2 has an essential function in suppressing GCR formation after chromosome breakage. Cells that harbor truncated Brca2 spontaneously incur GCRs and genomic DNA breaks during division. They exhibit hypersensitivity to DNA damage by interstrand cross-linkers, which even at low doses trigger aberrant genetic exchange between nonhomologous chromosomes. Therefore, genetic instability in Brca2-deficient cells results from the mutagenic processing of spontaneous or induced DNA damage into gross chromosomal rearrangements, providing a mechanistic basis for cancer predisposition.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas/genética , Silenciador del Gen , Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Translocación Genética , Animales , Anexina A5/metabolismo , Proteína BRCA2 , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Citometría de Flujo , Predisposición Genética a la Enfermedad , Etiquetado Corte-Fin in Situ , Cariotipificación , Hígado/embriología , Ratones , Mitomicina/farmacología , Mutagénesis , Recombinasa Rad51 , Recombinación GenéticaRESUMEN
Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas , Proteínas de Escherichia coli , Escherichia coli/genética , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Microscopía Electrónica , Procesamiento Proteico-Postraduccional , Recombinación GenéticaRESUMEN
The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Tolerancia a Radiación/genética , Rayos Ultravioleta , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN/química , ADN/metabolismo , Cartilla de ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Fenotipo , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.
Asunto(s)
Síndrome de Cockayne/genética , ADN Helicasas/química , Reparación del ADN , Adenosina Trifosfatasas/metabolismo , Células Cultivadas , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , Humanos , Cinética , Proteínas de Unión a Poli-ADP-Ribosa , ARN/biosíntesis , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas , ADN/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Recombinación Genética/genética , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Conformación de Ácido NucleicoRESUMEN
Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.
Asunto(s)
Síndrome de Cockayne/genética , ADN Helicasas/genética , Reparación del ADN , Mutación , Alelos , Aminoácidos , Animales , Línea Celular , Cricetinae , Enzimas Reparadoras del ADN , ADN Complementario , Humanos , Mutagénesis , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , ARN/análisis , Rayos UltravioletaRESUMEN
Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.
Asunto(s)
Síndrome de Cockayne/genética , ADN Helicasas/fisiología , Reparación del ADN , ARN Polimerasa II/metabolismo , Transcripción Genética , Secuencia de Consenso , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , Células HeLa , Humanos , Sustancias Macromoleculares , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas/metabolismo , Factores de TranscripciónRESUMEN
In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another. One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription. This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription-coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes. The mechanism of TCR in eukaryotes is still largely unknown. It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli. In the latter case, one protein, the transcription repair-coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed. While the E. coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins.
Asunto(s)
Síndrome de Cockayne/genética , Reparación del ADN , Transcripción Genética , Animales , Síndrome de Cockayne/metabolismo , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de TranscripciónRESUMEN
A mouse model for the nucleotide excision repair disorder Cockayne syndrome (CS) was generated by mimicking a truncation in the CSB(ERCC6) gene of a CS-B patient. CSB-deficient mice exhibit all of the CS repair characteristics: ultraviolet (UV) sensitivity, inactivation of transcription-coupled repair, unaffected global genome repair, and inability to resume RNA synthesis after UV exposure. Other CS features thought to involve the functioning of basal transcription/repair factor TFIIH, such as growth failure and neurologic dysfunction, are present in mild form. In contrast to the human syndrome, CSB-deficient mice show increased susceptibility to skin cancer. Our results demonstrate that transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers contributes to the prevention of carcinogenesis in mice. Further, they suggest that the lack of cancer predisposition in CS patients is attributable to a global genome repair process that in humans is more effective than in rodents.
Asunto(s)
Síndrome de Cockayne/genética , Reparación del ADN/fisiología , Neoplasias Cutáneas/genética , Transcripción Genética/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Síndrome de Cockayne/fisiopatología , ADN Helicasas/deficiencia , ADN Helicasas/genética , Reparación del ADN/efectos de la radiación , Enzimas Reparadoras del ADN , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutagénesis/fisiología , Trastornos por Fotosensibilidad/genética , Trastornos por Fotosensibilidad/fisiopatología , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Represoras/genética , Neoplasias Cutáneas/fisiopatología , Factores de Transcripción/genética , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.
Asunto(s)
Reparación del ADN/genética , Endonucleasas , Mitomicina/farmacología , Acetoxiacetilaminofluoreno/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Alelos , Animales , Células CHO , Células Cultivadas , Cricetinae , ADN/biosíntesis , ADN Complementario , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Metanosulfonato de Etilo/efectos adversos , Rayos gamma/efectos adversos , Prueba de Complementación Genética , Células HeLa , Humanos , Técnicas de Inmunoadsorción , Mutágenos/farmacología , Proteínas/genética , Proteínas/inmunología , ARN/biosíntesis , Transfección , Rayos Ultravioleta/efectos adversosRESUMEN
Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.
Asunto(s)
Síndrome de Cockayne/enzimología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Ubiquitinas/metabolismo , Rayos Ultravioleta , Línea Celular , Cisplatino/farmacología , Síndrome de Cockayne/genética , Daño del ADN , Reparación del ADN , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Fosforilación , ARN Polimerasa II/efectos de la radiación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Transcripción Genética , TransfecciónRESUMEN
A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.
Asunto(s)
Células CHO , Mitomicina/farmacología , Mutágenos/toxicidad , Adenina/análogos & derivados , Adenina/farmacología , Aminoacridinas/farmacología , Animales , Células CHO/efectos de los fármacos , Adhesión Celular , Línea Celular , Supervivencia Celular , Aberraciones Cromosómicas , Cricetinae , Replicación del ADN/efectos de la radiación , Prueba de Complementación Genética , Mutagénesis Sitio-Dirigida , Compuestos de Mostaza Nitrogenada/farmacología , Fenotipo , ARN/biosíntesis , ARN/efectos de la radiación , Tolerancia a Radiación , Intercambio de Cromátides Hermanas , Rayos UltravioletaRESUMEN
The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.
Asunto(s)
Adenosina Trifosfatasas , Proteínas de Ciclo Celular , Reparación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Secuencia de Bases , Genoma Fúngico , Datos de Secuencia Molecular , Dímeros de Pirimidina/metabolismo , Tolerancia a Radiación , Saccharomyces cerevisiae/efectos de la radiación , Transcripción Genética , Rayos Ultravioleta/efectos adversosRESUMEN
Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.
Asunto(s)
Proteínas de Ciclo Celular , Síndrome de Cockayne/genética , ADN Helicasas/genética , Reparación del ADN/genética , ADN de Hongos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Daño del ADN , Enzimas Reparadoras del ADN , ADN de Hongos/efectos de la radiación , Humanos , Datos de Secuencia Molecular , Proteínas de Unión a Poli-ADP-Ribosa , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/efectos de la radiación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética , Rayos UltravioletaRESUMEN
The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.