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1.
Nature ; 474(7350): 208-11, 2011 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-21654804

RESUMEN

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.


Asunto(s)
Modelos Animales de Enfermedad , Hepacivirus/fisiología , Hepatitis C/genética , Hepatitis C/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Adenoviridae/genética , Adenoviridae/fisiología , Animales , Anticuerpos Bloqueadores/inmunología , Antígenos CD/genética , Antígenos CD/metabolismo , Células Cultivadas , Claudina-1 , Genotipo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatocitos/citología , Humanos , Inmunización Pasiva , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Receptores Virales/genética , Receptores Virales/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Tetraspanina 28 , Transfección , Tropismo Viral
2.
Mol Ther ; 23(11): 1734-1747, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26281898

RESUMEN

The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Vectores Genéticos , Inmunoglobulina G/inmunología , Activación de Linfocitos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/metabolismo , Línea Celular Tumoral , Células HEK293 , Hepacivirus/inmunología , Humanos , Lentivirus , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Transporte de Proteínas , Receptores de IgG/metabolismo , Transducción Genética , Proteínas del Envoltorio Viral/inmunología
3.
Proc Natl Acad Sci U S A ; 109(24): 9499-504, 2012 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-22623528

RESUMEN

Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-Å resolution reveal that the epitope is a ß-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu(413) and Trp(420) on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn(415) on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Hepacivirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Epítopos/química , Epítopos/inmunología , Evasión Inmune , Modelos Animales , Datos de Secuencia Molecular , Pan troglodytes , Conformación Proteica , Vacunas Virales/inmunología
4.
J Virol ; 86(23): 13085-8, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22973046

RESUMEN

We have determined the crystal structure of the broadly neutralizing antibody (bnAb) AP33, bound to a peptide corresponding to hepatitis C virus (HCV) E2 envelope glycoprotein antigenic site 412 to 423. Comparison with bnAb HCV1 bound to the same epitope reveals a different angle of approach to the antigen by bnAb AP33 and slight variation in its ß-hairpin conformation of the epitope. These structures establish two different modes of binding to E2 that antibodies adopt to neutralize diverse HCV.


Asunto(s)
Anticuerpos Neutralizantes/química , Hepacivirus/genética , Modelos Moleculares , Conformación Proteica , Proteínas del Envoltorio Viral/química , Anticuerpos Neutralizantes/metabolismo , Epítopos/química , Epítopos/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo
5.
Nucleic Acids Res ; 35(5): 1589-600, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17289754

RESUMEN

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H-N-H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3' extensions. The enzyme has a distinct catalytic domain, which contains the H-N-H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H-N-H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H-N-H enzyme family.


Asunto(s)
Elementos Transponibles de ADN , Endodesoxirribonucleasas/química , Intrones , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Sitios de Unión , Catálisis , Roturas del ADN de Doble Cadena , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Dimerización , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Estructura Terciaria de Proteína , Zinc/metabolismo
6.
PLoS One ; 11(7): e0158674, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27387377

RESUMEN

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.


Asunto(s)
Bioimpresión , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Hepatocitos/efectos de los fármacos , Imagenología Tridimensional , Hígado/efectos de los fármacos , Albúminas/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Fluoroquinolonas/administración & dosificación , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Levofloxacino/administración & dosificación , Lipopolisacáridos/metabolismo , Hígado/metabolismo , Naftiridinas/administración & dosificación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo
7.
J Food Prot ; 68(3): 494-8, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15771172

RESUMEN

This study evaluated the efficacy of ozone, chlorine, and hydrogen peroxide to destroy Listeria monocytogenes planktonic cells and biofilms of two test strains, Scott A and 10403S. L. monocytogenes was sensitive to ozone (O3), chlorine, and hydrogen peroxide (H2O2). Planktonic cells of strain Scott A were completely destroyed by exposure to 0.25 ppm O3 (8.29-log reduction, CFU per milliliter). Ozone's destruction of Scott A increased when the concentration was increased, with complete elimination at 4.00 ppm O3 (8.07-log reduction, CFU per chip). A 16-fold increase in sanitizer concentration was required to destroy biofilm cells of L. monocytogenes versus planktonic cells of strain Scott A. Strain 10403S required an ozone concentration of 1.00 ppm to eliminate planktonic cells (8.16-log reduction, CFU per milliliter). Attached cells of the same strain were eliminated at a concentration of 4.00 ppm O3 (7.47-log reduction, CFU per chip). At 100 ppm chlorine at 20 degrees C, the number of planktonic cells of L. monocytogenes 10403S was reduced by 5.77 log CFU/ml after 5 min of exposure and by 6.49 log CFU/ml after 10 min of exposure. Biofilm cells were reduced by 5.79 log CFU per chip following exposure to 100 ppm chlorine at 20 degrees C for 5 min, with complete elimination (6.27 log CFU per chip) after exposure to 150 ppm at 20 degrees C for 1 min. A 3% H2O2 solution reduced the initial concentration of L. monocytogenes Scott A planktonic cells by 6.0 log CFU/ml after 10 min of exposure at 20 degrees C, and a 3.5% H2O2 solution reduced the planktonic population by 5.4 and 8.7 log CFU/ml (complete elimination) after 5 and 10 min of exposure at 20 degrees C, respectively. Exposure of cells grown as biofilms to 5% H2O2 resulted in a 4.14-log CFU per chip reduction after 10 min of exposure at 20 degrees C and in a 5.58-log CFU per chip reduction (complete elimination) after 15 min of exposure.


Asunto(s)
Antiinfecciosos Locales/farmacología , Biopelículas/crecimiento & desarrollo , Cloro/farmacología , Peróxido de Hidrógeno/farmacología , Listeria monocytogenes/fisiología , Ozono/farmacología , Recuento de Colonia Microbiana , Relación Dosis-Respuesta a Droga , Microbiología de Alimentos , Listeria monocytogenes/efectos de los fármacos , Temperatura , Factores de Tiempo
8.
Sci Transl Med ; 6(254): 254ra129, 2014 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-25232181

RESUMEN

In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. We demonstrate that three broadly nAbs-AR3A, AR3B, and AR4A-delivered with adeno-associated viral vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Hepatitis C Crónica/inmunología , Animales , Anticuerpos Neutralizantes/uso terapéutico , Células Cultivadas , Dependovirus/genética , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/terapia , Hepatocitos/virología , Humanos , Ratones
9.
Curr Biol ; 21(3): 243-8, 2011 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-21256016

RESUMEN

It is well understood how mobile introns home to allelic sites, but how they are stimulated to transpose to ectopic locations on an evolutionary timescale is unclear. Here we show that a group I intron can move to degenerate sites under oxidizing conditions. The phage T4 td intron endonuclease, I-TevI, is responsible for this infidelity. We demonstrate that I-TevI, which promotes mobility and is subject to autorepression and translational control, is also regulated posttranslationally by a redox mechanism. Redox regulation is exercised by a zinc finger (ZF) in a linker that connects the catalytic domain of I-TevI to the DNA binding domain. Four cysteines coordinate Zn(2+) in the ZF, which ensures that I-TevI cleaves its DNA substrate at a fixed distance, 23-25 nucleotides upstream of the intron insertion site. We show that the fidelity of I-TevI cleavage is controlled by redox-responsive Zn(2+) cycling. When the ZF is mutated, or after exposure of the wild-type I-TevI to H(2)O(2), intron homing to degenerate sites is increased, likely because of indiscriminate DNA cleavage. These results suggest a mechanism for rapid intron dispersal, joining recent descriptions of the activation of biomolecular processes by oxidative stress through cysteine chemistry.


Asunto(s)
Bacteriófago T4/genética , Endodesoxirribonucleasas/fisiología , Intrones , Estrés Oxidativo , Dedos de Zinc/genética , Zinc/metabolismo , Bacteriófago T4/enzimología , Secuencia de Bases , Sitios de Unión , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Regulación Viral de la Expresión Génica , Datos de Secuencia Molecular , Oxidación-Reducción , Análisis de Secuencia de ADN
10.
J Bacteriol ; 187(23): 7881-9, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16291661

RESUMEN

We describe a CCCH type of zinc finger domain in a replication protein A (RPA) homolog found in members of different lineages of the Euryarchaeota, a subdomain of Archaea. The zinc finger is characterized by CX(2)CX(8)CX(2)H, where X is any amino acid. Using MacRPA3, a representative of this new group of RPA in Methanosarcina acetivorans, we made two deletion mutants: a C-terminal deletion mutant lacking the zinc finger and an N-terminal deletion mutant containing the zinc finger domain. Whereas the N-terminal deletion mutant contained zinc at a level comparable to the wild-type protein level, the C-terminal deletion mutant was devoid of zinc. We further created four different mutants of MacRPA3 by replacing each of the four invariable amino acids in the zinc finger with alanine. Each single mutation at an invariable position resulted in a protein containing less than 35% of the zinc found in the wild-type protein. Circular dichroism spectra suggested that although the mutation at the first cysteine resulted in minor perturbation of protein structure, mutations at the other invariable positions led to larger structural changes. All proteins harboring a mutation at one of the invariable positions bound to single-stranded DNA weakly, and this translated into reduced capacity to stimulate DNA synthesis by M. acetivorans DNA polymerase BI. By subjecting the protein and its mutants to oxidizing and reducing conditions, we demonstrated that ssDNA binding by MacRPA3 may be regulated by redox through the zinc finger. Thus, the zinc finger modules in euryarchaeal RPA proteins may serve as a means by which the function of these proteins is regulated in the cell.


Asunto(s)
Proteínas Arqueales/genética , Euryarchaeota/genética , Proteína de Replicación A/genética , Dedos de Zinc/genética , Alanina , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/fisiología , Proteínas Portadoras , Replicación del ADN , ADN de Archaea/biosíntesis , ADN de Archaea/metabolismo , ADN de Cadena Simple/metabolismo , Euryarchaeota/química , Eliminación de Gen , Prueba de Complementación Genética , Iones/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión al ARN , Proteína de Replicación A/química , Proteína de Replicación A/fisiología , Alineación de Secuencia , Zinc/metabolismo
11.
J Biol Chem ; 280(51): 41852-63, 2005 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-16257971

RESUMEN

Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. Mac-RFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.


Asunto(s)
Methanosarcina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Genes Arqueales , Methanosarcina/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Homología de Secuencia de Aminoácido
12.
J Biol Chem ; 280(15): 15325-39, 2005 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-15671019

RESUMEN

The architecture of single-stranded DNA-binding proteins, which play key roles in DNA metabolism, is based on different combinations of the oligonucleotide/oligosaccharide binding (OB) fold. Whereas the polypeptide serving this function in bacteria contains one OB fold, the eukaryotic functional homolog comprises a complex of three proteins, each harboring at least one OB fold. Here we show that unlike these groups of organisms, the Euryarchaeota has exploited the potential in the OB fold to re-invent single-stranded DNA-binding proteins many times. However, the most common form is a protein with two OB folds and one zinc finger domain. We created several deletion mutants of this protein based on its conserved motifs, and from these structures functional chimeras were synthesized, supporting the hypothesis that gene duplication and recombination could lead to novel functional forms of single-stranded DNA-binding proteins. Biophysical studies showed that the orthologs of the two OB fold/one zinc finger replication protein A in Methanosarcina acetivorans and Methanopyrus kandleri exhibit two binding modes, wrapping and stretching of DNA. However, the ortholog in Ferroplasma acidarmanus possessed only the stretching mode. Most interestingly, a second single-stranded DNA-binding protein, FacRPA2, in this archaeon exhibited the wrapping mode. Domain analysis of this protein, which contains a single OB fold, showed that its architecture is similar to the functional homologs thought to be unique to the Crenarchaeotes. Most unexpectedly, genes coding for similar proteins were found in the genomes of eukaryotes, including humans. Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence.


Asunto(s)
ADN de Cadena Simple/metabolismo , Euryarchaeota/genética , Euryarchaeota/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anisotropía , Unión Competitiva , Fenómenos Biofísicos , Biofisica , Cromatografía en Gel , ADN/metabolismo , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Transferencia Resonante de Energía de Fluorescencia , Eliminación de Gen , Humanos , Cinética , Methanosarcina/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Filogenia , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Temperatura , Dedos de Zinc
13.
J Biol Chem ; 279(8): 6315-26, 2004 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-14676214

RESUMEN

Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination. We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans. RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain. Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers. Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M. acetivorans RPAs can act as a distinct single-stranded DNA-binding protein. Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M. acetivorans RPAs bind to as few as 10 single-stranded DNA bases. However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 +/- 0.28 nM, 173.6 +/- 105.17 nM, and 5.92 +/- 0.23 nM, for RPA1, RPA2, and RPA3, respectively. The architectures of the M. acetivorans RPAs are different from those of hitherto reported homologs. Thus, these proteins may represent novel forms of replication protein A. Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M. acetivorans DNA polymerase BI. Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon.


Asunto(s)
ADN de Cadena Simple/química , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Methanosarcina/metabolismo , Secuencia de Aminoácidos , Anisotropía , Cromatografía en Gel , Clonación Molecular , Cisteína/química , ADN/química , Reparación del ADN , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Datos de Secuencia Molecular , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Zinc/química , Dedos de Zinc
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