RESUMEN

An early and accurate diagnosis of Alzheimer's disease (AD) is required to initiate symptomatic treatment with currently approved drugs and will be of even greater importance if disease modifying compounds in development display a clinical effect. Protein profiles of human cerebrospinal fluid samples from AD patients (n=95) and population-based healthy controls (n=72) were analyzed by SELDI-TOF-MS in order to discover and characterize novel candidate biomarker combinations that differentiate AD patients from normal aging in this explorative study. Thirty candidate biomarkers (ROC AUC>0.7) were discovered that could differentiate patients with AD from healthy controls. Protein sequence determination and positive identification of 15 biomarkers revealed potential associations between the identified markers and AD pathogenesis. A multi-marker combination of five peaks could distinguish AD from healthy control individuals with high sensitivity (97%) and specificity (98%). The panel of five markers was tested on a blinded independent data set of 30 AD samples and 28 controls giving 100% sensitivity and 97% specificity. This novel panel of biomarkers could potentially be used to improve the accuracy of diagnosis of AD.

Asunto(s)

Enfermedad de Alzheimer/diagnóstico , Proteínas del Tejido Nervioso/sangre , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

BACKGROUND: An early and accurate diagnosis of Alzheimer's disease (AD) is important in order to initiate symptomatic treatment with currently approved drugs and will be of even greater importance with the advent of disease-modifying compounds. METHODS: Protein profiles of human cerebrospinal fluid samples from patients with AD (n = 85), frontotemporal dementia (n = 20), and healthy controls (n = 32) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to verify previously discovered biomarkers. RESULTS: We verified 15 protein biomarkers that were able to differentiate between AD and controls, and 7 of these 15 markers also differentiated AD from FTD. CONCLUSION: A panel of cerebrospinal fluid protein markers was verified by a proteomics technology which may potentially improve the accuracy of the AD diagnosis.

Asunto(s)

Envejecimiento/fisiología , Enfermedad de Alzheimer , Demencia/diagnóstico , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/fisiopatología , Biomarcadores , Cromogranina A/líquido cefalorraquídeo , Cistatina C , Cistatinas/líquido cefalorraquídeo , Diagnóstico Diferencial , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Ribonucleasa Pancreática/líquido cefalorraquídeo

RESUMEN

We previously demonstrated that 4.7 kDa and 4.4 kDa peptides are useful in diagnosing acute rejection in renal transplant recipients. The aim of this study was to characterize these polypeptides and assess their potential as biomarkers. The polypeptides were identified as human beta-Defensin-1 (4.7 kDa) and alpha-1-antichymotrypsin (4.4 kDa), by tandem mass spectrometry and ProteinChip immunoassay. The urinary abundance of both polypeptides, assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), revealed a reduction in beta-Defensin-1 while alpha-1-antichymotrypsin increased in patients with rejection (p < 0.05) compared with clinically stable transplants. The area under the curve (AUC) for the receiver operator characteristic (ROC) curve for the diagnosis of rejection for the ratio of both peptides combined was 0.912. Longitudinal analysis confirmed a reduction in beta-Defensin-1 with a reciprocal increase in alpha-1-antichymotrypsin as rejection developed. The difference in urinary beta-Defensin-1 levels quantified by radioimmunoassay was 176.8 +/- 122.3 pg/mL in stable patients compared with 83.2 +/- 52.2 pg/mL in patients with acute rejection, with an ROC AUC of 0.749 (p < 0.01). Immunohistochemistry (IHC) confirmed reduced beta-Defensin-1 expression in the renal parenchyma of patients experiencing acute rejection. In conclusion, the ratio of beta-Defensin-1 and alpha-1-antichymotrypsin excretion in the urine is a novel, potentially useful candidate biomarkers of acute rejection.

Asunto(s)

Rechazo de Injerto/orina , Trasplante de Riñón/patología , Péptidos/orina , Enfermedad Aguda , Biomarcadores/orina , Humanos , Inmunohistoquímica , Inmunosupresores/uso terapéutico , Trasplante de Riñón/inmunología , Peso Molecular , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trasplante Homólogo , alfa 1-Antiquimotripsina/orina , beta-Defensinas/orina

RESUMEN

BACKGROUND/AIMS: Amyloid beta (Abeta) is the principal component of senile plaques, one of the hallmarks of Alzheimer's disease (AD). Evidence is accumulating that soluble aggregates (oligomers) of Abeta are important in the pathogenesis of AD. METHODS: We compared three different methods for quantification of the 40 amino acid form of Abeta (Abeta40) in CSF, two based on antibodies [ELISA and surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) with antibody-coated arrays] and one based on direct binding of proteins to a protein array [SELDI-TOF and immobilized metal affinity [copper] (IMAC30)]. RESULTS: CSF Abeta40 concentration was only found to be significantly elevated in AD (127% of control levels; p=0.0095) using SELDI-TOF with IMAC30 arrays. CONCLUSIONS: These data suggest that the measured Abeta level in CSF may differ depending on whether antibody-based methods are used or not, possibly caused by epitope masking due to Abeta oligomerization or to binding of Abeta to carrier proteins.

Asunto(s)

Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Fragmentos de Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/análisis , Biomarcadores/líquido cefalorraquídeo , Líquido Cefalorraquídeo/química , Femenino , Humanos , Inmunoquímica/métodos , Masculino , Fragmentos de Péptidos/líquido cefalorraquídeo , Valores de Referencia , Reproducibilidad de los Resultados

RESUMEN

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.

Asunto(s)

Liasas de Carbono-Oxígeno/metabolismo , Ciclinas/metabolismo , Reparación del ADN/fisiología , Inhibidores Enzimáticos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Ciclo Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/metabolismo , Activación Enzimática , Endonucleasas de ADN Solapado , Humanos , Antígeno Nuclear de Célula en Proliferación/química

RESUMEN

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

Asunto(s)

ADN Polimerasa beta/metabolismo , Reparación del ADN , Afidicolina/farmacología , ADN/biosíntesis , ADN/química , ADN Polimerasa III/metabolismo , Reparación del ADN/efectos de los fármacos , Didesoxinucleósidos/farmacología , Humanos , Modelos Genéticos , Especificidad por Sustrato

RESUMEN

Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.

Asunto(s)

Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Inhibidores Enzimáticos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Supresoras de Tumor , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/metabolismo , Sustitución de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Sitios de Unión , Dominio Catalítico , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Cisteína , Escherichia coli , Células HeLa , Humanos , Cinética , Ligasas/metabolismo , Mutagénesis Sitio-Dirigida , Proteína NEDD8 , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Serina , Ubiquitina-Proteína Ligasas

RESUMEN

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Cullin , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas del Helminto/metabolismo , Proteínas I-kappa B , Péptido Sintasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP/genética , Proteínas del Helminto/genética , Humanos , Cinética , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Proteína NEDD8 , Fosforilación , Proteínas Ligasas SKP Cullina F-box , Alineación de Secuencia , Transfección , beta Catenina , Proteínas con Repetición de beta-Transducina

RESUMEN

Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) delta and epsilon to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be 32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol delta holoenzyme assembly, while PCNA or PCNA.pol delta complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol delta and stimulate DNA replication. The readdition of RF-C to the isolated PCNA.DNA complex did not further stimulate pol delta DNA synthesis. We conclude that pol delta holoenzyme consists of PCNA and pol delta core and that RF-C serves only to load PCNA clamp.

Asunto(s)

ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Animales , Bovinos , ADN/aislamiento & purificación , ADN Polimerasa III/aislamiento & purificación , Cartilla de ADN , Proteínas de Unión al ADN/aislamiento & purificación , Humanos , Cinética , Sustancias Macromoleculares , Antígenos de Histocompatibilidad Menor , Reacción en Cadena de la Polimerasa , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína de Replicación C , Especificidad por Sustrato , Timo/enzimología , Transfección

RESUMEN

Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V. N., and Fanning, E. (1997) J. Biol. Chem. 272, 6303-6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36). Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not. Complexes of p40.p37.p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O'Donnell, M. , and Hurwitz, J. (1997) J. Biol. Chem. 272, 18974-18981). In contrast, complex p38.p37.p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity. Although p38 was not required for ATPase activity, the activity of the p40-his.p38.p37. p36 complex was more salt-resistant than that of the p40-his.p37.p36 complex. The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA. Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ. Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C. In each case, the Lys of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase delta DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Sitios de Unión , Bovinos , ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/genética , Vectores Genéticos , Humanos , Hidrólisis , Antígenos de Histocompatibilidad Menor , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/metabolismo , Proteína de Replicación C , Spodoptera

RESUMEN

Replication factor C (RF-C), a complex of five subunits, is an essential eukaryotic protein involved in both DNA replication and DNA repair. To generate an easily accessible source of human RF-C for biochemical and genetic studies, we cloned the cDNAs of all five subunits into baculoviruses so that each subunit could be expressed both as a non-fused polypeptide and as an N-terminal His-tagged fusion (-his). Co-infection of insect cells with five baculoviruses encoding individual RF-C subunits (p140, p40, p38, p37, and p36) yielded a protein preparation active in two assays characteristic for authentic RF-C: stimulation of DNA polymerase delta DNA synthesis on singly primed single-stranded DNA template and formation of a complex of proliferating cell nuclear antigen with circular double-stranded DNA. Functional recombinant RF-C containing p40-his, p37-his, or p36-his was isolated using affinity resin. Active RF-C was reconstituted only by co-expression of all five subunits. A model for assembly of RF-C from individual subunits was derived from co-purification experiments performed with various combinations of His-tagged and non-fused subunits expressed by co-infection of insect cells with recombinant baculoviruses. p37 and p36 are proposed to form the first intermediate, which, upon addition of either p40 or p38, generates stable tertiary complexes: p40.p37.p36 and p38.p37.p36. The remaining fourth small subunit binds to the tertiary complex to form a quaternary complex p40.p38. p37.p36. Large subunit p140 binds last to form the five-subunit protein.

Asunto(s)

Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Homeodominio , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Baculoviridae , ADN Polimerasa III , ADN de Cadena Simple/metabolismo , Humanos , Sustancias Macromoleculares , Antígenos de Histocompatibilidad Menor , Peso Molecular , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes , Proteína de Replicación C , Relación Estructura-Actividad

RESUMEN

In order to study the effect of trimerization of proliferating cell nuclear antigen (PCNA) on its interaction with DNA polymerase (pol) delta and its loading onto DNA by replication factor C (RF-C) we have mutated a single tyrosine residue located at the subunit interface (Tyr114) to alanine. This mutation (Y114A) had a profound effect on PCNA, since it completely abolished trimer formation as seen by glycerol gradient sedimentation and native gel electrophoresis. Furthermore, the mutant protein was unable to stimulate DNA synthesis by pol delta and did not compete effectively with wild-type PCNA for pol delta, although it was able to oligomerize and could to some extent interact with subunits of functionally active PCNA. We thus conclude that PCNA molecules that are not part of a circular trimeric complex cannot interact with the pol delta core. furthermore, the mutant protein could not be loaded onto DNA by RF-C and did not compete with wild-type PCNA for loading onto DNA, indicating that PCNA trimerization may also be a prerequisite for its recognition by RF-C. The adverse effects caused by this single mutation suggest that trimerization of PCNA is essential for the monomers to keep their overall structure and that the structural changes imposed by trimerization are important for interaction with other proteins.

Asunto(s)

Proteínas de Homeodominio , Antígeno Nuclear de Célula en Proliferación/química , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Tirosina/química , Alanina/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/metabolismo , ADN Polimerasa III , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Histidina/metabolismo , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutación Puntual , Antígeno Nuclear de Célula en Proliferación/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Replicación C

RESUMEN

It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most to the inhibition of pol delta holoenzyme activity. p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA. Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.

Asunto(s)

Ciclinas/farmacología , Replicación del ADN/efectos de los fármacos , Proteínas de Homeodominio , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Unión Competitiva , Catálisis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN Polimerasa III , ADN Circular/biosíntesis , ADN Circular/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Antígenos de Histocompatibilidad Menor , Inhibidores de Proteínas Quinasas , Proteína de Replicación C , Moldes Genéticos

RESUMEN

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.

Asunto(s)

ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleósidos , Formicinas , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Nucleósidos de Pirimidina , ADN Polimerasa Dirigida por ARN/metabolismo , Composición de Base , Secuencia de Bases , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , Escherichia coli/enzimología , Transcriptasa Inversa del VIH , VIH-1/enzimología , Enlace de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Moldes Genéticos , Timo/enzimología

RESUMEN

To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.

Asunto(s)

ADN Circular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Replicación del ADN , ADN Circular/ultraestructura , ADN Polimerasa Dirigida por ADN/metabolismo , Mamíferos , Microscopía Electrónica , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/ultraestructura , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteína de Replicación C

RESUMEN

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.

Asunto(s)

Cisplatino/farmacología , Codón , ADN Polimerasa I/metabolismo , Fosfatos de Dinucleósidos/farmacología , Genes ras , Animales , Secuencia de Bases , Bovinos , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Timo/enzimología

RESUMEN

DNA polymerase delta requires proliferating cell nuclear antigen and replication factor C to form a holoenzyme efficient in DNA synthesis. We have analyzed three different aspects of calf thymus DNA polymerase delta holoenzyme: (i) analysis of pausing during DNA synthesis, (ii) replication of double-stranded DNA in the absence of additional factors, and (iii) replication of double-stranded DNA in the presence of the two known replicative DNA helicases from simian virus 40 and bovine papilloma virus. DNA polymerase delta holoenzyme replicated primed single-stranded DNA at a rate of 100-300 nucleotides/min, partially overcoming multiple pause sites on DNA. While Escherichia coli single-strand DNA binding protein helped DNA polymerase delta pass through pause sites, the DNA polymerase delta itself appeared to dissociate from the template in the absence of synthesis or when encountering pause sites. Proliferating cell nuclear antigen likely remained on the template. DNA polymerase delta holoenzyme could perform limited strand displacement synthesis on double-stranded gapped circular DNA, and this reaction was not stimulated either by replication protein A or by E. coli single-strand DNA binding protein. DNA polymerase delta holoenzyme could efficiently cooperate with replicative DNA helicases from simian virus 40 (large T antigen) and bovine papilloma virus 1 (protein E1) in replication through double-stranded DNA in a reaction that required replication protein A or E. coli single-strand DNA binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Bacteriófago T4 , Papillomavirus Bovino 1 , ADN Polimerasa III , Replicación del ADN , ADN Circular/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A , Virus 40 de los Simios , Moldes Genéticos

RESUMEN

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.

Asunto(s)

Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Homeodominio , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Bacteriófago T4 , ADN/efectos de la radiación , ADN Ligasas/metabolismo , ADN Polimerasa II , ADN Polimerasa III , Exodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado , Humanos , Antígenos de Histocompatibilidad Menor , Proteína de Replicación A , Proteína de Replicación C , Rayos Ultravioleta

RESUMEN

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Asunto(s)

Daño del ADN , Reparación del ADN , Replicación del ADN , Endonucleasas , Animales , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa II , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Células HeLa , Humanos , Mamíferos , Plásmidos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo A

RESUMEN

Highly selective affinity labeling of a DNA-polymerase alpha-primase complex from human placenta by o-formylphenyl esters of ATP, ADP and AMP was performed in a two-step procedure in which a substrate analog attached to the active center was elongated by radioactive ATP. If the covalent attachment is performed in the presence of poly(dT) template, the ATP esters modify selectively the delta subunit of the complex. If poly(dT) is added after the covalent binding of the reagent, both delta and gamma subunits become labeled. With the o-formylphenyl ester of AMP the delta-subunit is modified. The ADP ester modifies both the delta and gamma subunit in the presence and absence of template. It is shown that formylphenyl ester of ATP is not the substrate in the reaction of elongation catalyzed by primase. The data obtained suggest the binding site of initiating substrate to be located in the region of contact of the two subunits of primase. The role of the template in the formation of the active site is discussed.

Asunto(s)

Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad , ADN Polimerasa Dirigida por ADN , Placenta/enzimología , ARN Nucleotidiltransferasas , ADN Primasa , Humanos , Oligonucleótidos/biosíntesis , Poli A/metabolismo , ARN Nucleotidiltransferasas/metabolismo