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Journal articles on the topic 'ADN polymerase I'

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Published: 4 June 2021
Last updated: 3 February 2022

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1

Pan, Junhua, Vikram N. Vakharia, and Yizhi Jane Tao. "The structure of a birnavirus polymerase reveals a distinct active site topology." Proceedings of the National Academy of Sciences 104, no. 18 (April 24, 2007): 7385–90. http://dx.doi.org/10.1073/pnas.0611599104.

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Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.
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2

Jurado-Orejuela, Diana, Claudia Paredes-Amaya, and Gerardo Libreros-Zúñiga. "Technical validation of a polymerase chain reaction for Chlamydia trachomatis detection." Salud Uninorte 32, no. 3 (August 5, 2021): 398–410. http://dx.doi.org/10.14482/sun.32.3.9807.

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Objetivo: Implementar una técnica de PCR (Reacción en cadena de la polimerasa) para la detección de Chlamydia trachomatis. Materiales y Métodos: Se realizó un estudio experimental en el que se estandarizaron las condiciones de PCR (Concentración de MgCl2 , Taq polimerasa y temperatura de alineamiento de cebadores) para la amplificación de una región de 201pb del plásmido de C. trachomatis con los cebadores CtP1 5´-TAGTAACTGCCACTTCATCA-3´ y CtP2 5´- TTCCCCTTGTAATTCGTTGC-3. Se realizaron diluciones seriadas del ADN de C. trachomatis ATCC VR885D para determinar la sensibilidad analítica. La especificidad analítica de los cebadores se determinó con ADN de diferentes microorganismos patógenos y comensales del tracto urogenital. Se determinó la variabilidad intra e interensayo de la PCR sobre triplicados de diferentes muestras de ADN. Resultados: Las condiciones de amplificación fueron 94°C/4 min, seguido de 40 ciclos a 94°C/1 min, 56°C/1 min y 72°C/1.5 min y extensión final de 72°C/4 min. La concentración óptima de MgCl2 fue 1.5 mM y de ADN polimerasa 1U. La sensibilidad analítica de la prueba fue 4.8 x10-15g/mL de ADN equivalentes a 160 cuerpos elementales de C. trachomatis. La especificidad analítica fue 100% y la variabilidad intra e interensayo mostraron reproducibilidad de la PCR. Conclusiones: Los datos sugieren que esta PCR puede emplearse con fines diagnósticos. Se requieren estudios adicionales para la evaluación clínica de esta prueba.
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3

De Witte, J. C., I. Deblauwe, Gill De Deken, R. De Deken, M. Madder, and Rudolf Meiswinkel. "Puces à ADN comme outil d'identification moléculaire pour Culicoides." Revue d’élevage et de médecine vétérinaire des pays tropicaux 62, no. 2-4 (February 1, 2009): 144. http://dx.doi.org/10.19182/remvt.10054.

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A DNA microarray test based on internal transcribed spacer 1 (ITS1) genotype expression was developed to identify Culicoides species of Northwestern Europe belonging to the Culicoides Obsoletus complex. The assay was designed so as to allow interpretation by the naked eye. False positive and false nega­tive results were eliminated. The need for expensive laboratory equipment and reagents was kept as low as possible, making the technique affordable and feasible for any diagnostic laboratory with polymerase chain reaction (PCR) facilities. The microarray test could be validated through the three ringtests organised by Medreonet. Use of this microarray can improve monitoring adult and immature Culicoides species.
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4

CERVANTES GONZALES, Jorge Luis,. "Obtención de ácido desoxirribonucleico (ADN) útil para análisis genético, a partir de uñas recortadas." Revista Medica Herediana 14, no. 4 (April 5, 2013): 230. http://dx.doi.org/10.20453/rmh.v14i4.712.

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Obtaining deoxyribonucleic acid (DNA) is the starting point for most genetic analysis. Nails are an accessible source of DNA. The present communication reports the successful extraction of genomic DNA from fresh nails, as well as from nails collected a month before the extraction. Amplification in two different regions of the human beta-globin gene was achieved by of the polymerase chain reaction. The described method, is a simple, non invasive method. Nail clipping material may be considered a convenient material for genetic analysis. (Rev Med Hered 2003; 14:230-233).
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5

PITEL, F., and J. RIQUET. "Les marqueurs anonymes et la détection de leur polymorphisme." INRAE Productions Animales 13, HS (December 22, 2000): 45–53. http://dx.doi.org/10.20870/productions-animales.2000.13.hs.3810.

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Les marqueurs génétiques les plus utilisés actuellement en génétique animale sont présentés, sans que soient développées dans le détail toutes les techniques mises en oeuvre. Nous distinguons les marqueurs utilisés avant la PCR (Polymerase Chain Reaction) et ceux qui sont employés depuis. Les marqueurs actuels sont également présentés en deux groupes : ceux qui sont utilisés pour une approche globale du génome et ceux que l’on emploie dans des approches ponctuelles, en distinguant la mise en évidence d’un polymorphisme et son exploitation à grande échelle. Nous évoquons enfin les SNP (Single Nucleotide Polymorphism), qui seront probablement des marqueurs très utilisés dans l’avenir grâce à la technologie des ’puces à ADN’.
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6

Hernández F., Javier, Leonardo Mariño, Martha L. Orozco C., and Javier Narvaez V. "Uso de la Reacción en Cadena de la Polimerasa para caracterizar aislamientos nativos de Bacillus thuringiensis." Corpoica Ciencia y Tecnología Agropecuaria 2, no. 1 (July 31, 1997): 1. http://dx.doi.org/10.21930/rcta.vol2_num1_art:156.

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<p>En este estudio se estandarizó una metodología para la caracterización molecular de cepas nativas de Bacillus thuringiensis, la cual se basó en la amplificación de los genes cry mediante la Reacción en Cadena de la Polimerasa (PCR). Se utilizaron cuatro mezclas de oligonucleótidos: dos Generales (I y II), los cuales reconocen genes de las familias cry1, cry2, cry3, cry4 y cry1Ia, y dos Específicos (A y B), que identifican los genes de la familia cry1 (cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca y cry1Da). La calidad y concentración del ADN bacteriano influyó sobre la especificidad y concentración de los productos obtenidos mediante la amplificación de los genes cry. La calidad del ADN purificado a través del método rápido reportado por M. He et al. (Nucleic Ac. Res. 18:1660, 1990) permitió una amplificación eficiente. Para realizar la PCR en condiciones óptimas, se utilizó una mezcla de reacción con un volumen final de 20 µl, la cual contenía 0,4 µM de cada oligonucleótido, 1X PCR buffer (50 mM KCL, wmM Tris-HCl, pH 8,3 y 3,0 mM MgCl 2), 200 µM de cada dNTP y 1U de Taq-ADN-polimerasa, además de 10-100 y 300- 500 ng de ADN bacterial para las mezclas de los oligonucleótidos Generales y Específicos, respectivamente. El programa de amplificación incluyó 30 ciclos de desnaturalización a 94°C, hibridación a 53°C y síntesis a 72°C durante 20 segundos cada uno. La metodología estandarizada se puede utilizar rutinariamente para amplificar los genes cry procedentes de aislamientos nativos de B. thuringiensis, lo cual permite clasificarlos y seleccionarlos de una manera rápida y precisa de acuerdo con su actividad biológica y potencial biotecnológico; adicionalmente, como paso previo de los ensayos de toxicidad contra diversas especies de insectos plaga de interés agrícola.</p><p><strong><br /></strong></p><p><strong>Utilization of Polymerase Chain Reaction for Characterization of Native Isolates of Bacillus thuringiensis</strong></p><p>A methodology based on Polymerase Chain Reaction (PCR) techniques was standardized for the molecular characterization of cry genes in Bacillus thuringiensis. Four oligonucleotides mixes (primers) were used: two General (I and II) -which recognize the genes families cry1, cry2, cry3, cry4 and cry1Ia-, and two Specific (A and B) which recognize the genes family cry1 (cry1Aa, crylAb, cry1Ac, cry1Ba, cry1Ca and cry1Da). The quality of bacterial DNA influenced the amplification product specificity and concentration. The quality of the DNA purified by M. He et al. fast method (Nucleic Ac. Res. 18:1660, 1990) allowed an efficient amplification. The optimum conditions for PCR were achieved using a mixture reaction with a final volume of 20 µL which contained 0.4 µM of each primer, 1X PCR buffer (50 mM KCL, 10mM Tris-HCI, pH 8.3 and 3.0 mM MgCI,), 200 µM dNTP's, 1 U Taq-DNA-polymerase, 10-100 ng of bacterial DNA for the General mixtures and 300-500 ng of bacterial DNA for the Specific mixtures. The amplification program included 30 cycles as follows: denaturation at 94°C, annealing at 53°C and synthesis at 72°C during 20 seconds each one. The standardized methodology could be used routinely in the cry genes amplification of native isolates of B. thuringiensis for the classification, rapid and precise selection according to the potential biological activity as a previous step to toxicity trials against diverse insect pests of agriculture interest.</p>
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7

Letzel, Tobias, Egbert Mundt, and Alexander E. Gorbalenya. "Evidence for functional significance of the permuted C motif in Co2+-stimulated RNA-dependent RNA polymerase of infectious bursal disease virus." Journal of General Virology 88, no. 10 (October 1, 2007): 2824–33. http://dx.doi.org/10.1099/vir.0.82890-0.

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Segment B of bisegmented infectious bursal disease virus (IBDV) encodes virus protein 1 (VP1), possessing RNA-dependent RNA polymerase (RdRp) activity. This multidomain protein includes an RdRp domain with a non-canonical order of three sequence motifs forming the active site: C–A–B. The A–B–C order of the motifs, as found in RdRps of the majority of viruses, was converted by relocation (permutation) of motif C to a C–A–B order. Due to the unusual location and unproven significance, the motif was named ‘C?’. This motif includes an Ala–Asp–Asn tripeptide that replaces the C motif Gly–Asp–Asp sequence, widely considered a hallmark of RdRps. In this study, functional significance of the C? motif was investigated by using purified His-tagged VP1 mutants with either a double replacement (ADN to GDD) or two single-site mutants (ADD or GDN). All mutants showed a significant reduction of RdRp activity in vitro, in comparison to that of VP1. Only the least-affected GDN mutant gave rise to viable, albeit partially impaired, progeny using a reverse-genetics system. Experiments performed to investigate whether the C motif was implicated in the control of metal dependence revealed that, compared with Mn2+ and Mg2+, Co2+ stimulated RdRp unconventionally. No activity was observed in the presence of several divalent cations. Of two Co2+ salts with Cl− and anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 μM Co2+, an increase in IBDV progeny yield was observed. The obtained results provide evidence that the unusual Co2+ dependence of the IBDV RdRp might be linked to the permuted organization of the motif.
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8

Peters, Inga, Kai Gebauer, Faranaz Atschekzei, Joerg Hennenlotter, Mario W. Kramer, Wolfgang Traenkenschuh, Axel S. Merseburger, Markus Kuczyk, and Juergen Serth. "CpG-island methylation of GATA-family members GATA3 and GATA5 in renal cell carcinoma and association with clinicopathological parameters and progression-free survival." Journal of Clinical Oncology 30, no. 5_suppl (February 10, 2012): 370. http://dx.doi.org/10.1200/jco.2012.30.5_suppl.370.

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370 Background: Transcriptional inactivation and CGI methylation of GATA3 and −5 has been reported to be involved in mammary carcinoma, pancreatic cancer, colorectal and gastric carcinogenesis. A recent study demonstrated that a loss of GATA-3 expression due to partially methylation silencing in several renal cell carcinoma (RCC) patients. We quantitatively investigated GATA3 and −5 CGI methylation in RCC and analyzed its association with clinical characteristics as well as progression free survival of patients. Methods: Methylation data were obtained from a quantitative methylation-specific polymerase chain reaction assay (QMSP) for both genes. We investigated 108 RCC and 77 paired tissue samples as well as six RCC cell lines. Statistical analyses were carried out using the paired t-test for matched tumor (TU) and adjacent normal (adN) samples, logistic regression for comparisons of independent samples and cox regression for survival analysis. Results: In paired samples we found a significant higher methylation in TU compared to adN for GATA3 (P=0.007) and for GATA5 (P=3.6*10−9) for all RCCs. GATA5 showed also strong correlations between methylation and status of metastasis (P=0.05) and advanced (pT≥3 and/or N+, M+) tumor samples compared to localized (pT≤2, N0, M0) tumors (P=4.7*10−9). A decreased progression free survival in cox proportional hazard model analysis could be demonstrated for patients with a high GATA5 methylation (P=0.0006, HR=6.5) and a trend could also be seen for GATA3 methylated patients (P=0.06). Conclusions: GATA3 and −5 were identified to demonstrate tumor-specific CGI hypermethylation in renal cell cancer patients. The association of GATA5 CGI methylation with metastasis, advanced disease and progression free survival of patients indicates that epigenetic alterations of both genes are involved in renal cell carcinogenesis. GATA5 methylation could serve as a biomarker for tumor progression. Further prospective and functional investigations are necessary to clarify whether CGI methylation of GATA family members can provide independent information for future clinical management of patients with RCC.
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9

Neyra, Carlos D., Marilyn R. Suarez, Eddie D. Cueva, Henri Bailón, and Ericson L. Gutierrez. "Identificación genética de recién nacidos en Perú: un estudio piloto." Revista Chilena de Pediatría 90, no. 1 (February 19, 2019): 26. http://dx.doi.org/10.32641/rchped.v90i1.730.

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Objetivo: Determinar la factibilidad de la identificación genética a un grupo de recién nacidos provenientes de un hospital público de Lima-Perú. Material y Método: Estudio descriptivo de corte transversal, realizado por Registro de Identificación y Estado Civil de Perú, en recién nacidos vivos y sus respectivas madres, provenientes del Hospital Carlos Lanfranco La Hoz (Puente Piedra-Lima) durante el mes de enero del 2015. Las muestras fueron colectadas en tarjetas FTA (Fast Technology for Analysis of nucleic acids) que permitieron un análisis directo por PCR (Polymerase Chain Reaction) y electroforesis capilar de 21 marcadores genéticos de tipo STR (Short Tandem Repeats), incluyendo el marcador amelogenina para la determinación del sexo. Resultados: Se incluyeron un total de 44 madres y 45 recién nacidos (existió un parto gemelar). La probabilidad de maternidad fue mayor al 99.9% en todos los casos. No se encontraron dificultades en la toma de muestra, ni en el transporte del material. El material biológico obtenido fue suficiente para la obtención de ADN para realizar la identificación del recién nacido. Conclusiones: El procedimiento de identificación genética fue factible de realizar en este hospital. Se identificaron etapas del proceso que podrían mejorarse para la posible aplicación de este procedimiento a una mayor escala en el Perú.
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10

Soro, Kolotcholohofolo, Thérèse Atcham Agneroh, and Kouakou Théodore Kouadio. "Identification of eggplant (Solanum melongena) as a new host of begomovirus Pepper yellow vein Mali virus in Côte d’Ivoire." Journal of Applied Biosciences 157 (January 31, 2021): 16153–60. http://dx.doi.org/10.35759/jabs.157.1.

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Objective: Eggplant (Solanum melongena) is one of the important vegetables in Africa and Asia. Begomoviruses are emerging plant viruses that cause significant losses. However, there is little research on begomoviruses infecting eggplant. Therefore, this study aimed at identifying begomoviruses infecting eggplant. Methodology and results: Six samples of virus-like infected eggplants were collected in Ferkessedougou in the North of Cote d’Ivoire. The molecular tests Polymerase Chain Reaction (PCR) and Rolling Circle Amplification (RCA) were performed on the samples. One sample tested positive by PCR and RCA while the five others were negative by PCR for begomoviruses. Products from both tests were sequenced to get partial sequence of begomovirus Pepper yellow vein Mali virus (PepYVMLV) from PCR and two full genome components DNA A and DNA B of PepYVMLV from RCA. The sequences were released in Genbank. Conclusion and application of findings: This study has done the molecular characterization of the complete two genome sequence components DNA A and DNA B of Pepper yellow vein Mali on eggplant. Agro-infection of eggplants with the two components could reveal actual specific symptoms which are caused by PepYVMLV on eggplant. This could help opens possibilities of engineering resistant eggplant to PepYMLV. Keywords: Eggplant, begomovirus, Pepper yellow vein Mali virus, new host, Cote d’Ivoire RESUME Objectif: L’aubergine (Solanum melongena) est l’un des légumes les plus importants en Afrique et en Asie. Les begomovirus sont des virus émergents qui causent de pertes importantes. Toutefois, il y a très peu de recherches sur les begomovirus de l’aubergine. Ainsi, cette étude visait à l’identification des begomovirus infectant l’aubergine. Méthodologie et résultats: Nous avons collecté 6 échantillons d’aubergine à Ferkéssédougou au Nord de la Côte d’Ivoire, parmi des plants d’aubergine qui présentaient des symptômes de type viral. Les tests moléculaires Polymerase Chain Reaction (PCR) et Rolling Circle Amplification (RCA) ont été réalisés sur les échantillons. Un échantillon a été positif à la fois à la PCR et la RCA alors que les 5 autres étaient négatifs à la PCR pour les begomovirus. Le séquençage des produits de la PCR a donné une séquence partielle du Soro et al., J. Appl. Biosci. 2021 Identification of eggplant (Solanum melongena) as a new host of begomovirus Pepper yellow vein Mali virus in Côte d’Ivoire 16154 begomovirus Pepper yellow vein Mali virus (PepYVMLV). Les produits issus de la RCA ont donné des séquences des composants ADN A et ADN B de PepYVMLV qui ont été publiées dans le Genbank. Conclusion et application des résultats: Notre étude a effectué la caractérisation moléculaire des deux sequences complètes des composantes DNA A et DNA B du génome complet du Pepper yellow vein Mali virus sur l’aubergine. L’agro-infectioon des aubergines avec les deux composantes pourrait révéler les symptômes spécifiques réels qui sont causés par le PepYVMLV sur l’aubergine. Cela pourrait ouvrir des possibilités de mise en place de variétés d’aubergines résistantes au PepYMLV. Mots-clés: Aubergine, begomovirus, Pepper yellow vein Mali virus, nouvelle plante hôte, Côte d’Ivoi
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11

Lukashev, Alexander N., Olga E. Ivanova, Tatiana P. Eremeeva, and Richard D. Iggo. "Evidence of frequent recombination among human adenoviruses." Journal of General Virology 89, no. 2 (February 1, 2008): 380–88. http://dx.doi.org/10.1099/vir.0.83057-0.

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Genome stability is a prerequisite for the production and use of adenoviruses for therapy of genetic diseases and cancer. To test the premise that the adenoviral genome is stable, the phylogenetic relationships of 16 adenovirus C (AdC) field isolates were studied in four genome regions: hexon, fiber, polymerase and E1A. The phylogenetic relationships in the fiber gene concurred with those in the hexon region. In contrast, the non-structural regions had marks of frequent recombination, to the point that an isolate of one serotype could contain non-structural proteins that were identical to the genes from a different serotype. Our results suggest that recombination among circulating adenoviruses is very frequent and plays an important role in shaping the phylogenetic relationships of adenovirus genomes. Analysis of the available complete genome sequences of AdB, AdC and AdD species showed that recombination shuffles genome fragments within a species, but not between species. One of the AdC field isolates possessed the fiber gene of AdC type 6, but a hexon gene that was distinct from all AdC serotypes. This strain could not be typed unambiguously in a neutralization test and might represent a novel serotype of AdC. Comparison of the right end (nt 18838–33452) of this isolate with that of the ATCC Ad6 strain showed clear evidence of multiple recombination events.
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12

Golubov, Andrey, Priti Maheshwari, Andriy Bilichak, and Igor Kovalchuk. "New in vitro dna polymerase activity and fidelity assay reveals age-dependent changes in Arabidopsis Thaliana." International Journal of Plant Biology 2, no. 1 (June 20, 2011): 7. http://dx.doi.org/10.4081/pb.2011.e7.

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DNA polymerase is an enzyme that adds nucleotides to the growing DNA chain during replication and DNA repair. DNA polymerase activity and fidelity are important characteristics that reflect the ability of DNA polymerase to add nucleotides and then proofread newly synthesized DNA. We have developed a protocol allowing analysis of polymerase activity and fidelity using crude Arabidopsis thaliana plant extracts. It is based on the ability of DNA polymerases in the extract to elongate the fluorescently labelled primer annealed to a short complementary template. For analysis, fluorescently labelled products were separated on a denaturing polyacrylamide gel and visualized using a high performance blot imager. Analysis of tissue prepared from 5-, 12- and 21-day-old Arabidopsis plants showed an age-dependent decrease in polymerase activity, an increase in polymerase fidelity and a tendency to an increase in exo- (endo) nucleolytic activity.
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13

Choi, Jeong Jin, Jae-Geun Song, Ki Hoon Nam, Jong Il Lee, Heejin Bae, Gun A. Kim, Younguk Sun, and Suk-Tae Kwon. "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase." Applied and Environmental Microbiology 74, no. 21 (September 12, 2008): 6563–69. http://dx.doi.org/10.1128/aem.00624-08.

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ABSTRACT 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 λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 � 10−6) than Taq DNA polymerase (11.98 � 10−6). Uniquely, Neq DNA polymerase also amplified λ 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.
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14

Hałas, A., A. Ciesielski, and J. Zuk. "Involvement of the essential yeast DNA polymerases in induced gene conversion." Acta Biochimica Polonica 46, no. 4 (December 31, 1999): 862–72. http://dx.doi.org/10.18388/abp.1999_4107.

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In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.
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15

Tuteja, Renu, Abulaish Ansari, and Virander Singh Chauhan. "Emerging Functions of Transcription Factors in Malaria Parasite." Journal of Biomedicine and Biotechnology 2011 (2011): 1–6. http://dx.doi.org/10.1155/2011/461979.

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Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs) are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression.Plasmodium falciparumis responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control inP. falciparumsomehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite.
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McDonald, John P., Agnès Tissier, Ekaterina G. Frank, Shigenori Iwai, Fumio Hanaoka, and Roger Woodgate. "DNA polymerase iota and related Rad30–like enzymes." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 356, no. 1405 (January 29, 2001): 53–60. http://dx.doi.org/10.1098/rstb.2000.0748.

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Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so–called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase η and defects in the protein lead to the xeroderma pigmentosum variant (XP–V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol ι. Based upon in vitro studies, it appears that Pol ι has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error–prone DNA polymerase.
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Egorova, Tatiana, Ekaterina Shuvalova, Sabina Mukba, Alexey Shuvalov, Peter Kolosov, and Elena Alkalaeva. "Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides." International Journal of Molecular Sciences 22, no. 10 (May 14, 2021): 5186. http://dx.doi.org/10.3390/ijms22105186.

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Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.
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Téllez Gil, Luis Eduardo, Elvia María Michelli Viña, Diana Estela Callejas Monsalve, Mike Contreras Colmenares, María Eugenia Cavazza Porro, and María Correnti de Plata. "Presencia de lesiones preinvasoras e invasoras de cérvix, relación con el virus papiloma humano y factores epidemiológicos en Mérida, Venezuela." QhaliKay. Revista de Ciencias de la Salud ISSN: 2588-0608 2, no. 2 (May 1, 2018): 92. http://dx.doi.org/10.33936/qhalikay.v2i2.1664.

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Las lesiones de cérvix se han asociado a infección por Virus Papiloma Humano (VPH). 300 mujeres mayores de quince años que acudieron al Hospital Universitario de Los Andes (HULA), fueron estudiadas para identificar lesiones, detectar y tipificar VPH, y determinar factores asociados. Se realizó citología, colposcopia, cepillados cervicales utilizando (DNA collection device Digene®) y biopsias en los casos pertinentes. Se aisló el ADN mediante (QIAamp DNA Mini Kit QIAGEN®), siendo cuantificado y almacenado a -20 ºC. Se detectó VPH por Reacción en Cadena de la Polimerasa (PCR) de regiones L1 y E6/E7. La genotipificación por PCR anidada múltiple E6/E7, C. trachomatis se detectó por PCR. El VPH se detectó en 35 % (105) muestras, 88,46 % (92/105) fueron positivas para al menos uno de los genotipos evaluados. VPHAR se encontraron en 97,82 %, (90/92), VPH18 en 82 % (74/90), VPH16 en 44 % (40/90). 56,52 % (52/92) correspondieron a infecciones múltiples, VPH18/16 (20/52) fue la más frecuente. C. trachomatis se detectó en 9 % (27/300) pacientes. La citología mostró cambios sugestivos de infección en solo 16,35 % de las pacientes VPH positivas. 17/18 biopsias sugirieron infección viral y fueron positivas para VPH AR por biología molecular (94,44 %). La colposcopia sugirió infección viral en 46,15 %. El 66,34 % de pacientes fueron menores de 35 años. Se encontró relación estadísticamente no significativa entre infección por VPH, número de parejas sexuales, coinfección con C. trachomatis y hábito tabáquico. Estos resultados muestran elevada frecuencia de infección por VPH AR, asociada a factores epidemiológicos, cuyo diagnóstico certero y tratamiento oportuno son claves en la prevención de su transmisión y del desarrollo de lesiones en cérvix. Palabras clave: Cáncer cervical, virus papiloma humano, reacción en cadena de la polimerasa. Abstract Cervical lesions have been associated with infection by Human Papilloma Virus (HPV). Three hundred women older than 15 years old who attended at the Hospital Universidad de Los Andes (HULA), were studied to identify lesions, detect and typify HPV, and determine associated factors. Cytology, colposcopy, cervical brushing using (DNA collection device Digene®) and biopsies were performed in the pertinent cases. DNA was isolated by (QIAamp DNA Mini Kit QIAGEN®), being quantified and stored at -20 ° C. HPV was detected by Polymerase Chain Reaction (PCR) of regions L1 and E6 / E7. The genotyping by multiple nested PCR E6 / E7, C. trachomatis was detected by PCR. HPV was detected in 35% (105) samples, 88.46% (92/105) were positive for at least one of the genotypes evaluated. VPHAR were found in 97.82% (90/92), HPV18 in 82% (74/90), HPV16 in 44% (40/90). 56.52% (52/92) corresponded to multiple infections, HPV18 / 16 (20/52) was the most frequent. C. trachomatis was detected in 9% (27/300) patients. The cytology showed changes suggestive of infection in only 16.35% of the HPV positive patients. 17/18 biopsies suggested viral infection and were positive for ARV HPV by molecular biology (94.44%). Colposcopy suggested viral infection in 46.15%. 66.34% of patients were under 35 years old. A statistically non-significant relationship was found between HPV infection, number of sexual partners, coinfection with C. trachomatis and smoking habit. These results show high frequency of infection by HPV AR, associated with epidemiological factors, whose accurate diagnosis and timely treatment are key in the prevention of its transmission and the development of lesions in the cervix. Keywords: Cervical cancer, human papilloma virus, polymerase chain reaction.
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Pospiech, Helmut, and Juhani E. Syväoja. "DNA Polymerase e - More Than a Polymerase." Scientific World JOURNAL 3 (2003): 87–104. http://dx.doi.org/10.1100/tsw.2003.08.

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This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.
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20

Steitz, Thomas A., and Y. Whitney Yin. "Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1441 (January 29, 2004): 17–23. http://dx.doi.org/10.1098/rstb.2003.1374.

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The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.
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21

Wild, Gary E., Patrizia Papalia, Mark J. Ropeleski, Julio Faria, and Alan BR Thomson. "Applications of Recombinant Dna Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part B: Eukaryotic Gene Transcription and Post-Transcripional Rna Processing." Canadian Journal of Gastroenterology 14, no. 4 (2000): 283–92. http://dx.doi.org/10.1155/2000/385327.

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The transcription of DNA into RNA is the primary level at which gene expression is controlled in eukaryotic cells. Eukaryotic gene transcription involves several different RNA polymerases that interact with a host of transcription factors to initiate transcription. Genes that encode proteins are transcribed into messenger RNA (mRNA) by RNA polymerase II. Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) are transcribed by RNA polymerase I and III, respectively. The production of each mRNA in human cells involves complex interactions of proteins (ie, trans-acting factors) with specific sequences on the DNA (ie, cis-acting elements). Cis-acting elements are short base sequences adjacent to or within a particular gene. While the regulation of transcription is a pivotal step in the control of gene expression, a variety of molecular events, collectively known as ’RNA processing’ add an additional level of control of gene expression in eukaryotic cells.
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SARAFIANOS, Stefanos G., Ulrich KORTZ, Michael T. POPE, and Mukund J. MODAK. "Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft." Biochemical Journal 319, no. 2 (October 15, 1996): 619–26. http://dx.doi.org/10.1042/bj3190619.

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The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12 O36]16- (polyoxometalate I) and [(O3PCH2PO3)4 W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 µM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme–polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules.
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23

Abu Al-Soud, Waleed, and Peter Rådström. "Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples." Applied and Environmental Microbiology 64, no. 10 (October 1, 1998): 3748–53. http://dx.doi.org/10.1128/aem.64.10.3748-3753.1998.

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ABSTRACT The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase fromThermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaqGold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, andTfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima(Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTthfrom Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.
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24

Rajagopal, Deepa, Robert W. Maul, Amalendu Ghosh, Tirtha Chakraborty, Ahmed Amine Khamlichi, Ranjan Sen, and Patricia J. Gearhart. "Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation." Journal of Experimental Medicine 206, no. 6 (May 11, 2009): 1237–44. http://dx.doi.org/10.1084/jem.20082514.

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Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.
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25

Korkhin, Yakov, Ulug M. Unligil, Otis Littlefield, Pamlea J. Nelson, David I. Stuart, Paul B. Sigler, Stephen D. Bell, and Nicola G. A. Abrescia. "Evolution of Complex RNA Polymerases: The Complete Archaeal RNA Polymerase Structure." PLoS Biology 7, no. 5 (May 5, 2009): e1000102. http://dx.doi.org/10.1371/journal.pbio.1000102.

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26

Swain, Umakanta, Gilgi Friedlander, Urmila Sehrawat, Avital Sarusi-Portuguez, Ron Rotkopf, Charlotte Ebert, Tamar Paz-Elizur, et al. "TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer." International Journal of Molecular Sciences 22, no. 13 (June 28, 2021): 6957. http://dx.doi.org/10.3390/ijms22136957.

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TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.
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27

Sen, S., S. Mukhopadhyay, J. Wetzel, and T. K. Biswas. "Characterization of the mitochondrial DNA polymerase from Saccharomyces cerevisiae." Acta Biochimica Polonica 41, no. 1 (March 31, 1994): 79–86. http://dx.doi.org/10.18388/abp.1994_4777.

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The mitochondrial DNA (mtDNA) polymerase was isolated from a protease-deficient yeast strain (PY2), and purified about 3000 fold by a column chromatography on phosphocellulose, heparin-agarose, and single-stranded DNA cellulose. The purified polymerase was characterized with respect to optimal nucleotide concentrations, template-primer specificity and sensitivity to some inhibitors. These results were compared with the nuclear DNA polymerase I activity. Both polymerases showed similar requirement of deoxynucleotide concentrations (Km < 1 microM), and highest activity with poly(dA-dT) template. However, the mtDNA polymerase was more sensitive to ddTTP, EtBr and Mn2+ inhibition in comparison to the nuclear DNA polymerase I. The mtDNA polymerase did not need ATP as an energy source for in vitro DNA synthesis. This mtDNA polymerase preparation also showed 3'-->5' exonuclease activity.
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28

Somesh, Baggavalli P., James Reid, Wei-Feng Liu, T. Max M. Søgaard, Hediye Erdjument-Bromage, Paul Tempst, and Jesper Q. Svejstrup. "Multiple Mechanisms Confining RNA Polymerase II Ubiquitylation to Polymerases Undergoing Transcriptional Arrest." Cell 121, no. 6 (June 2005): 913–23. http://dx.doi.org/10.1016/j.cell.2005.04.010.

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29

Hałas, A., Z. Policińska, H. Baranowska, and W. J. Jachymczyk. "The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae." Acta Biochimica Polonica 46, no. 2 (June 30, 1999): 289–98. http://dx.doi.org/10.18388/abp.1999_4162.

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We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.
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30

Priya, V. Sri Vajra, Hare Krishna Roy, N. jyothi, and N. Lakshmi Prasanthi. "Polymers in Drug Delivery Technology, Types of Polymers and Applications." Scholars Academic Journal of Pharmacy 5, no. 7 (July 2016): 305–8. http://dx.doi.org/10.21276/sajp.2016.5.7.7.

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31

Sharland, M., J. Hodgson, E. G. Davies, J. Booth, and S. Jeffery. "Enteroviral pharyngitis diagnosed by reverse transcriptase-polymerase chain reaction." Archives of Disease in Childhood 74, no. 5 (May 1, 1996): 462–63. http://dx.doi.org/10.1136/adc.74.5.462.

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32

Sendy, Bandar, David J. Lee, Stephen J. W. Busby, and Jack A. Bryant. "RNA polymerase supply and flux through the lac operon in Escherichia coli." Philosophical Transactions of the Royal Society B: Biological Sciences 371, no. 1707 (November 5, 2016): 20160080. http://dx.doi.org/10.1098/rstb.2016.0080.

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Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli . By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase. This article is part of the themed issue ‘The new bacteriology’.
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33

Forterre, Patrick, Christiane Eue, Mouldy Sioud, and Abdellah Hamal. "Studies on DNA polymerases and topoisomerases in archaebacteria." Canadian Journal of Microbiology 35, no. 1 (January 1, 1989): 228–33. http://dx.doi.org/10.1139/m89-035.

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We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.Key words: archaebacteria, DNA topoisomerases, DNA polymerases, DNA topology, gyrase.
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34

Shevelev, Igor V., Kristijan Ramadan, and Ulrich Hubscher. "The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy." Scientific World JOURNAL 2 (2002): 275–81. http://dx.doi.org/10.1100/tsw.2002.99.

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Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.
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35

Syväoja, Juhani E. "DNA polymerase epsilon: The latest member in the family of mammalian DNA polymerases." BioEssays 12, no. 11 (November 1990): 533–36. http://dx.doi.org/10.1002/bies.950121106.

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36

Groppi, A., J. Begueret, and A. Iron. "Improved methods for genotype determination of human alcohol dehydrogenase (ADH) at ADH 2 and ADH 3 loci by using polymerase chain reaction-directed mutagenesis." Clinical Chemistry 36, no. 10 (October 1, 1990): 1765–68. http://dx.doi.org/10.1093/clinchem/36.10.1765.

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Abstract The human gene for producing alcohol dehydrogenase (ADH; EC 1.1.1.1) is polymorphic at ADH 2 and ADH 3 loci. Until now, the study of this polymorphism required liver biopsy or allele-specific radioactive probes. We have used directed mutagenesis by the polymerase chain reaction (PCR) to amplify and analyze the genotype of ADH 2 and ADH 3 loci. Thus, we could determine easily and unambiguously the complete genotype at these two loci by using a microsample of blood and restriction fragment length polymorphism after DNA amplification by PCR.
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37

Narita, M., Y. Matsuzono, O. Itakura, S. Yamada, and T. Togashi. "Analysis of mycoplasmal pleural effusion by the polymerase chain reaction." Archives of Disease in Childhood 78, no. 1 (January 1, 1998): 67–69. http://dx.doi.org/10.1136/adc.78.1.67.

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38

Hosoya, M. "Diagnosis of group A coxsackieviral infection using polymerase chain reaction." Archives of Disease in Childhood 87, no. 4 (October 1, 2002): 316–19. http://dx.doi.org/10.1136/adc.87.4.316.

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39

Makioka, A., B. Stavros, J. T. Ellis, and A. M. Johnson. "Detection and characterization of DNA polymerase activity in Toxoplasma gondii." Parasitology 107, no. 2 (August 1993): 135–39. http://dx.doi.org/10.1017/s0031182000067238.

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SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.
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Sudarwanto, Mirnawati, Surachmi Setiyaningsih, and Harsi Dewantari Kusumaningrum. "Isolation of Campylobacter from Poultry Carcasses using Conventional and Polymerase Chain Reaction Methods." Jurnal Teknologi dan Industri Pangan 24, no. 1 (June 2013): 27–32. http://dx.doi.org/10.6066/jtip.2013.24.1.27.

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41

Davydov, Evgeny, Irina Gaponova, Georgy Pariiskii, Tatyana Pokholok, and Gennady Zaikov. "Reactivity of Polymers on Exposure to Nitrogen Dioxide." Chemistry & Chemical Technology 4, no. 4 (December 15, 2010): 281–90. http://dx.doi.org/10.23939/chcht04.04.281.

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The mechanism of reactions of nitrogen dioxide with polymers of different classes is considered. In reactions with carbon-chain polymers at 298 K, nitrogen dioxide can only add to extrinsic double bonds that are formed in the synthesis of the polymers. The mechanism of reactions of nitrogen dioxide with polymers of different classes is considered. In reactions with carbon-chain polymers at 298 K, nitrogen dioxide can only add to extrinsic double bonds that are formed in the synthesis of the polymers. These reactions resulted in dinitro compounds and nitro nitrites. At elevated temperatures, carbonyl and hydroxyl groups are formed in these polymers along with nitration products. Active participants of NO2 reactions with rubbers are double bonds converting into nitroalkyl and alkyl radicals initiating then free radical conversions of these polymers. Polymers containing amide, urethane and imide groups are rather sensitive to NO2. These materials undergo essential changes in the chemical structure with formation of stable nitrogen-containing radicals. The reactions of nitrogen dioxide provide a simple method of the spin-labeled polymer preparation.
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42

Zwierzchowski, L., W. Niedbalski, and D. Kleczkowska. "Effect of prolactin, progesterone, pregnancy and lactation on DNA synthesis and DNA polymerase activities in rabbit mammary gland." Journal of Endocrinology 114, no. 1 (July 1987): 139–45. http://dx.doi.org/10.1677/joe.0.1140139.

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ABSTRACT DNA synthesis and DNA polymerase-α, -β and -γ activities in the rabbit mammary gland were studied during hormone-directed cellular growth. It was found that during pregnancy, early lactation and after injection of prolactin, changes in the activity of DNA polymerase-α paralleled the rate of mammary gland DNA synthesis. It was also found that the amount of polymerase-α activity bound to isolated chromatin depended on the physiological state of the animal. During pregnancy and early lactation changes in the activity of chromatin-bound enzyme correlated directly with the rate of DNA synthesis (r = 0·83). Moreover, in virgin rabbits treated with prolactin the activity of chromatin-bound DNA polymerase-α increased markedly at the same time as the DNA-synthetic rate increased. No correlation of the DNA-synthetic rate was found with the activity of soluble (cytosolic) DNA polymerase-α or with the activity of soluble or chromatin-bound DNA polymerases-β and -γ. On the basis of these results it is suggested that in the developing mammary gland both the activity and cellular distribution of DNA polymerase-α might be subject to hormonal regulation. J. Endocr. (1987) 114, 139–145
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43

Nakamura, Akiyoshi, Taiki Nemoto, Isao Tanaka, and Min Yao. "Structural analysis of tRNA(His) guanylyltransferase comlexed with tRNA." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1816. http://dx.doi.org/10.1107/s2053273314081844.

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tRNA(His) guanylyltransferase (Thg1) of eukaryote adds a guanylate to the 5' end of immature or incorrectly processed tRNAs (3'-5' polymerization) by three reaction steps: adenylylation; guanylylation and dephosphorylation. This additional guanylate provides the major identity element for histidyl-tRNA synthetase to recognize its cognate substrate tRNA(His) and differentiates tRNA(His) from the pool of tRNAs present in the cell (1). Previous studies indicate that Thg1 is a structural homolog of canonical 5'-3' polymerases in the catalytic core with no obvious conservation of the amino acid sequence(2). However, the substrate binding of Thg1 is unclear and requires information on the three-dimensional structure in complex with tRNA. In this study, we determined the crystal structures of Thg1 from Candida albicans (CaThg1) in tRNA-bound (CaThg1-tRNA), ATP-bound (CaThg1-ATP), and GTP-bound (CaThg1-GTP) form, and elucidated how Thg1 functions as a reverse polymerase to add nucleotide(3). The crystal structures of CaThg1-tRNA complex shows that two tRNAs are bound to tetrameric Thg1 in parallel orientation which is consistent with SAXS (Small angle X-ray scattering) and gel filtration analysis. One tRNA interacts with three monomers for its positioning, anticodon recognition, and catalytic activation. The end of the acceptor stem and the anticodon loop are both recognized by the same sub-domain belonging to the different monomers. Moreover, the structural comparison of Thg1-tRNA with canonical 5'-3' polymerase shows that the domain architecture of Thg1 is reversed to that of canonical 5'-3' polymerase.
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44

Reynaud, Claude-Agnès, Frédéric Delbos, Ahmad Faili, Quentin Guéranger, Said Aoufouchi, and Jean-Claude Weill. "Competitive repair pathways in immunoglobulin gene hypermutation." Philosophical Transactions of the Royal Society B: Biological Sciences 364, no. 1517 (November 14, 2008): 613–19. http://dx.doi.org/10.1098/rstb.2008.0206.

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This review focuses on the contribution of translesion DNA polymerases to immunoglobulin gene hypermutation, in particular on the roles of DNA polymerase eta (Polη) in the generation of mutations at A/T bases from the initial cytosine-targeted activation-induced cytidine deaminase (AID)-mediated deamination event, and of Polκ, an enzyme of the same polymerase family, used as a substitute when Polη is absent. The proposition that the UNG uracil glycosylase and the MSH2–MSH6 mismatch recognition complex are two competitive rather than alternative pathways in the processing of uracils generated by AID is further discussed.
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45

Szalewska-Pałasz, Agnieszka. "Properties of Escherichia coli RNA polymerase from a strain devoid of the stringent response alarmone ppGpp." Acta Biochimica Polonica 55, no. 2 (June 14, 2008): 317–23. http://dx.doi.org/10.18388/abp.2008_3078.

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The stringent response alarmone guanosine tetraphosphate (ppGpp) affects transcription from many promoters. ppGpp binds directly to the transcription enzyme of Escherichia coli, RNA polymerase. Analysis of the crystal structure of RNA polymerase with ppGpp suggested that binding of this nucleotide may result in some conformational or post-translational alterations to the enzyme. These changes might affect in vitro performance of the enzyme. Here, a comparison of the in vitro properties of RNA polymerases isolated from wild type and ppGpp-deficient bacteria shows that both enzymes do not differ in i) transcription activity of various promoters (e.g. sigma(70)-rrnB P1, lambdapL, T7A1), ii) response to ppGpp, iii) promoter-RNA polymerase open complex stability. Thus, it may be concluded that ppGpp present in the bacterial cell prior to purification of the RNA polymerase does not result in the alterations to the enzyme that could be permanent and affect its in vitro transcription capacity.
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46

Wright, G. E. "Nucleotide probes of DNA polymerases." Acta Biochimica Polonica 43, no. 1 (March 31, 1996): 115–24. http://dx.doi.org/10.18388/abp.1996_4522.

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The modified nucleotides, N2-(p-n-butylphenyl)dGTP and 2-(p-n-butylanilino) dATP and related compounds have been developed as inhibitor-probes of B family DNA polymerases. Synthetic approaches to these compounds are summarized. The nucleotides are potent, non-substrate inhibitors of DNA polymerase a. In contrast, they inhibit other members of the family with less potency but act as substrates for these enzymes. Modelling of the inhibitor: enzyme binding mechanism has been done based on the known structure of E. coli DNA polymerase I, and site-directed mutagenesis experiments to evaluate this mechanism are proposed.
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47

Szafranski, Przemyslaw, and W. Jerzy Smagowicz. "Relative Affinities of Nucleotide Substrates for the Yeast tRNA Gene Transcription Complex." Zeitschrift für Naturforschung C 47, no. 3-4 (April 1, 1992): 320–22. http://dx.doi.org/10.1515/znc-1992-3-426.

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Abstract Apparent Michaelis constants for nucleotides in transcription of yeast tRN Agene by hom ologous RNA polymerase III with auxiliary protein factors, were found to be remarkably higher in initiation than in elongation of RNA chain. This supports presumptions regarding topological similarities between catalytic centers of bacterial and eukaryotic RNA polymerases.
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48

Ikegami, H., M. Fukuda, Y. Kawaguchi, Y. Fujioka, and T. Ogihara. "Detection of Pstl RFLP in human ADA by the polymerase chain reaction." Nucleic Acids Research 19, no. 19 (1991): 5448. http://dx.doi.org/10.1093/nar/19.19.5448.

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49

Wu, Jiqin, Guoliang Lu, Bo Zhang, and Peng Gong. "Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation." Journal of Virology 89, no. 1 (October 15, 2014): 249–61. http://dx.doi.org/10.1128/jvi.02085-14.

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ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.
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50

Saribasak, Huseyin, Robert W. Maul, Zheng Cao, William W. Yang, Dominik Schenten, Sven Kracker, and Patricia J. Gearhart. "DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions." Journal of Experimental Medicine 209, no. 6 (May 21, 2012): 1075–81. http://dx.doi.org/10.1084/jem.20112234.

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Low-fidelity DNA polymerases introduce nucleotide substitutions in immunoglobulin variable regions during somatic hypermutation. Although DNA polymerase (pol) η is the major low-fidelity polymerase, other DNA polymerases may also contribute. Existing data are contradictory as to whether pol ζ is involved. We reasoned that the presence of pol η may mask the contribution of pol ζ, and therefore we generated mice deficient for pol η and heterozygous for pol ζ. The frequency and spectra of hypermutation was unaltered between Polζ+/− Polη−/− and Polζ+/+ Polη−/− clones. However, there was a decrease in tandem double-base substitutions in Polζ+/− Polη−/− cells compared with Polζ+/+ Polη−/− cells, suggesting that pol ζ generates tandem mutations. Contiguous mutations are consistent with the biochemical property of pol ζ to extend a mismatch with a second mutation. The presence of this unique signature implies that pol ζ contributes to mutational synthesis in vivo. Additionally, data on tandem mutations from wild type, Polζ+/−, Polζ−/−, Ung−/−, Msh2−/−, Msh6−/−, and Ung−/− Msh2−/− clones suggest that pol ζ may function in the MSH2–MSH6 pathway.
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