RESUMEN
Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.
Asunto(s)
Escherichia coli , Uracil-ADN Glicosidasa , Escherichia coli/metabolismo , Mutación , Uracil-ADN Glicosidasa/química , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismoRESUMEN
Transcriptional activation domain (TAD) in virion protein 16 (VP16) of herpes simplex virus does not have any globular structure, yet exhibits a potent transcriptional activity. In order to probe the structural basis for the transcriptional activity of VP16 TAD, we have used NMR spectroscopy to investigate its detailed structural features. Results show that an unbound VP16 TAD is not merely "unstructured" but contains four short motifs (residues 424-433, 442-446, 465-467 and 472-479) with transient structural order. Pre-structured motifs in other intrinsically unfolded proteins (IUPs) were shown to be critically involved in target protein binding. The 472-479 motif was previously shown to bind to hTAF(II)31, whereas the hTAF(II)31-binding ability of other motifs found in this study has not been addressed. The VP16 TAD represents another IUP whose prestructured motifs mediate promiscuous binding to various target proteins.
Asunto(s)
Proteína Vmw65 de Virus del Herpes Simple/química , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Pliegue de Proteína , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Proteína Vmw65 de Virus del Herpes Simple/fisiología , Humanos , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Hypoxia-inducible factor 1alpha (HIF1alpha) is a transcription factor that plays a key role in the adaptation of cells to low oxygen stress and oxygen homeostasis. The oxygen-dependent degradation (ODD) domain of HIF1alpha responsible for the negative regulation of HIF1alpha in normoxia is intrinsically unfolded. Here, we carried out the backbone (1)H, (15)N, and (13)C resonance assignment of a proteolysis-resistant fragment (residues 404-477) in the HIF1alpha ODD domain using NMR spectroscopy. About 98% (344/352) of all the (1)HN, (15)N, (13)Calpha, (13)Cbeta, and (13)CO resonances were unambiguously assigned. The results will be useful for further investigation of the structural and dynamic states of the HIF1alpha ODD domain and its interaction with binding partners.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Oxígeno/metabolismo , Animales , Hipoxia de la CélulaRESUMEN
The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced lambda DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10(-6)) than Taq DNA polymerase (11.98 x 10(-6)). Uniquely, Neq DNA polymerase also amplified lambda DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nanoarchaeota/enzimología , Reacción en Cadena de la Polimerasa/métodos , Bacteriófago lambda/genética , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Hipoxantina/metabolismo , Peso Molecular , Especificidad por Sustrato , Uracilo/metabolismoRESUMEN
Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.
Asunto(s)
Archaea/enzimología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Thermococcus/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/química , Estabilidad de Enzimas , Escherichia coli/genética , Genes Bacterianos , Semivida , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Temperatura , Thermococcus/genéticaRESUMEN
Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.