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Polymerase substrate specificity

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https://www.readbyqxmd.com/read/28641560/functional-and-catalytic-characterization-of-the-detoxifying-enzyme-haloalkane-dehalogenase-from-rhizobium-leguminosarum
#1
Nikolaos Georgakis, Evangelia Chronopoulou, Maria Anna Gad, Dimitrios Skliros, Rodica Efrose, Emmanouil Flemetakis, Nikolaos E Labrou
Haloalkanes are acutely toxic to animals and humans and their toxic effects are mainly observed in the liver, kidneys and central nervous system. In the present work, the haloalkane dehalogenase from Rhizobium leguminosarum bv. trifolii (DrlA) was characterized. Reverse transcription polymerase chain reaction analysis and enzyme activity assays revealed that the DrlA gene expression in R. leguminosarum bv. trifolii is induced by 1,2-dibromoethane (1,2-DBE) during the early exponential phase. The gene of the enzyme was isolated, cloned and expressed in E...
June 20, 2017: Protein and Peptide Letters
https://www.readbyqxmd.com/read/28630124/the-dna-repair-repertoire-of-mycobacterium-smegmatis-fena-includes-the-incision-of-dna-5-flaps-and-the-removal-of-5-adenylylated-products-of-aborted-nick-ligation
#2
Maria Loressa Uson, Shreya Ghosh, Stewart Shuman
We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5' -flap endonuclease homologous to the 5' -exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' -single-strand is eliminated to create a 5' -tailed duplex, FenA incision shifts to between the second and third nucleotides...
June 19, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28629775/8-oxo-7-8-dihydroguanine-friend-and-foe-epigenetic-like-regulator-versus-initiator-of-mutagenesis
#3
REVIEW
Aaron M Fleming, Cynthia J Burrows
A high flux of reactive oxygen species during oxidative stress results in oxidative modification of cellular components including DNA. Oxidative DNA "damage" to the heterocyclic bases is considered deleterious because polymerases may incorrectly read the modifications causing mutations. A prominent member in this class is the oxidized guanine base 8-oxo-7,8-dihydroguanine (OG) that is moderately mutagenic effecting G→T transversion mutations. Recent reports have identified that formation of OG in G-rich regulatory elements in the promoters of the VEGF, TNFα, and SIRT1 genes can increase transcription via activation of the base excision repair (BER) pathway...
June 9, 2017: DNA Repair
https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#4
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28622802/rapid-multiplex-dna-amplification-on-an-inexpensive-microdevice-for-human-identification-via-short-tandem-repeat-analysis
#5
Jacquelyn A DuVall, Delphine Le Roux, Brandon L Thompson, Christopher Birch, Daniel A Nelson, Jingyi Li, Daniel L Mills, An-Chi Tsuei, Martin G Ensenberger, Cindy Sprecher, Douglas R Storts, Brian E Root, James P Landers
Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis...
August 8, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28588250/an-atypical-phosphodiesterase-capable-of-degrading-haloalkyl-phosphate-diesters-from-sphingobium-sp-strain-tcm1
#6
Katsumasa Abe, Naoko Mukai, Yuka Morooka, Takeshi Makino, Kenji Oshima, Shouji Takahashi, Yoshio Kera
Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters...
June 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28570559/analysis-of-dna-polymerase-%C3%AE-function-in-meiotic-recombination-immunoglobulin-class-switching-and-dna-damage-tolerance
#7
Kei-Ichi Takata, Shelley Reh, Matthew J Yousefzadeh, Maciej J Zelazowski, Sarita Bhetawal, David Trono, Megan G Lowery, Maria Sandoval, Yoko Takata, Yue Lu, Kevin Lin, Jianjun Shen, Donna F Kusewitt, Kevin M McBride, Francesca Cole, Richard D Wood
DNA polymerase ν (pol ν), encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν-defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology...
June 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28559253/nucleotide-substrate-specificity-of-hepatitis-c-analogs-for-human-mitochondrial-rna-polymerase
#8
Maryam Ehteshami, Longhu Zhou, Sheida Amiralaei, Jadd R Shelton, Jong Hyun Cho, Hongwang Zhang, Hao Li, Xiao Lu, Tugba Ozturk, Richard Stanton, Franck Amblard, Tamara R McBrayer, Steven J Coats, Raymond F Schinazi
Nucleoside analog inhibitors (NAI) are an important class of antiviral agents. Although highly effective, some NAI with anti-hepatitis C virus (HCV) activity can cause toxicity presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT enzyme was studied in order to explore structure-activity relationships that can facilitate the identification of non-toxic NAI. These finding have important implications for all anti-RNA virus NAI development...
May 30, 2017: Antimicrobial Agents and Chemotherapy
https://www.readbyqxmd.com/read/28538144/probing-the-potential-role-of-non-b-dna-structures-at-yeast-meiosis-specific-dna-double-strand-breaks
#9
Rucha Kshirsagar, Krishnendu Khan, Mamata V Joshi, Ramakrishna V Hosur, K Muniyappa
A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif...
May 23, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28528003/acinetobacter-baumannii-k11-and-k83-capsular-polysaccharides-have-the-same-6-deoxy-l-talose-containing-pentasaccharide-k-units-but-different-linkages-between-the-k-units
#10
Johanna J Kenyon, Alexander S Shashkov, Sof'ya N Senchenkova, Mikhail M Shneider, Bin Liu, Anastasiya V Popova, Nikolay P Arbatsky, Konstantin A Miroshnikov, Lei Wang, Yuriy A Knirel, Ruth M Hall
Acinetobacter baumannii produces a variety of capsular polysaccharides (CPS) via genes located at the chromosomal K locus and some KL gene clusters include genes for the synthesis of specific sugars. The structures of K11 and K83 CPS produced by isolates LUH5545 and LUH5538, which carry related KL11a and KL83 gene clusters, respectively, were established by sugar analysis and one- and two-dimensional (1)H and (13)C NMR spectroscopy. Both CPS contain l-rhamnose (l-Rha) and 6-deoxy-l-talose (l-6dTal), and both KL gene clusters include genes for dTDP-l-Rhap synthesis and a tle (talose epimerase) gene encoding an epimerase that converts dTDP-l-Rhap to dTDP-l-6dTalp...
May 17, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28524231/comparison-of-pre-ribosomal-rna-processing-pathways-in-yeast-plant-and-human-cells-focus-on-coordinated-action-of-endo-and-exoribonucleases
#11
REVIEW
Rafal Tomecki, Pawel J Sikorski, Monika Zakrzewska-Placzek
Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs...
May 19, 2017: FEBS Letters
https://www.readbyqxmd.com/read/28514164/the-nonbulky-dna-lesions-spiroiminodihydantoin-and-5-guanidinohydantoin-significantly-block-human-rna-polymerase-ii-elongation-in-vitro
#12
Marina Kolbanovskiy, Moinuddin A Chowdhury, Aditi Nadkarni, Suse Broyde, Nicholas E Geacintov, David A Scicchitano, Vladimir Shafirovich
The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II...
June 7, 2017: Biochemistry
https://www.readbyqxmd.com/read/28502712/ligation-mediated-pcr-with-a-back-to-back-adapter-reduces-amplification-bias-resulting-from-variations-in-gc-content
#13
Satoru Ishihara, Naoe Kotomura, Naoki Yamamoto, Hiroshi Ochiai
Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence...
May 11, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28474789/functional-characterization-and-tissue-expression-of-marmoset-cytochrome-p450-2e1
#14
Shotaro Uehara, Yasuhiro Uno, Etsuko Tomioka, Takashi Inoue, Erika Sasaki, Hiroshi Yamazaki
Common marmosets (Callithrix jacchus) have attracted increasing attention as a useful small non-human primate model in preclinical research. However, studies on marmoset cytochrome P450 (P450) 2E enzyme have scarcely been conducted. In this study, the full-length cDNA encoding P450 2E1 enzyme was isolated from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR). Marmoset P450 2E1 amino acid sequences were highly identical (>88%) to those of cynomolgus monkey and human P450 2E1 enzymes...
May 5, 2017: Biopharmaceutics & Drug Disposition
https://www.readbyqxmd.com/read/28469419/an-insight-into-structure-function-and-expression-analysis-of-3-hydroxy-3-methylglutaryl-coa-reductase-of-cymbopogon-winterianus
#15
Kamalakshi Devi, Lochana Patar, Mahendra K Modi, Priyabrata Sen
Citronella (Cymbopogon winterianus) is one of the richest sources of high-value isoprenoid aromatic compounds used as flavour, fragrance, and therapeutic elements. These isoprenoid compounds are synthesized by 2 independent pathways: mevalonate pathway and 2-C-methyl-d-erythritol-4-phosphate pathway. Evidence suggests that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a rate-controlling enzyme for the synthesis of variety of isoprenoids. This study reports the isolation, characterization, and tissue-specific expression analysis of HMGR from citronella...
2017: Bioinformatics and Biology Insights
https://www.readbyqxmd.com/read/28463692/synergy-of-substrate-conductivity-and-intermittent-electrical-stimulation-towards-osteogenic-differentiation-of-human-mesenchymal-stem-cells
#16
K Ravikumar, Sunil Kumar Boda, Bikramjit Basu
Human Mesenchymal Stem cells (hMSCs) have the unique potential to differentiate into multiple cell types. Depending on the cellular microenvironment (physical and biochemical cues), hMSCs can be directed to differentiate into osteogenic, chondrogenic, myogenic and adipogenic lineages. Among the strategies available to direct stem cell fate processes, electrical stimulation based approach has been extensively investigated in recent studies. In the present study, the conducting Hydroxyapatite-CaTiO3 (HA-CT) composites are used as electroconductive platforms to support the differentiation of hMSCs, in vitro...
March 30, 2017: Bioelectrochemistry
https://www.readbyqxmd.com/read/28450579/quantitative-polymerase-chain-reaction-analysis-of-the-mouse-cyp2c-subfamily-and-characterization-of-their-tissue-distribution
#17
Joan P Graves, Artiom Gruzdev, J Alyce Bradbury, Laura M DeGraff, Matthew L Edin, Darryl C Zeldin
The cytochrome P450 superfamily encompasses a diverse group of enzymes that catalyze the oxidation of various substrates. CYP2C proteins detoxify xenobiotics and metabolize endogenous lipids such as arachidonic acid to bioactive eicosanoids. We report new methods and results for the quantitative polymerase reaction (qPCR) analysis for the 15 members of the mouse Cyp2c subfamily (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c65, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69 and Cyp2c70)...
April 27, 2017: Drug Metabolism and Disposition: the Biological Fate of Chemicals
https://www.readbyqxmd.com/read/28395772/an-electrochemical-aptasensor-for-multiplex-antibiotics-detection-using-y-shaped-dna-based-metal-ions-encoded-probes-with-nmof-substrate-and-csrp-target-triggered-amplification-strategy
#18
Meng Chen, Ning Gan, Tianhua Li, Ye Wang, Qing Xu, Yinji Chen
An effective electrochemical aptasensor has been developed for the detection of multiplex antibiotics using Y-shaped DNA probes. These probes-based metal ions encoded the nanoscale metal-organic frameworks (NMOF) as a substrate, and circular strand-replacement DNA polymerization (CSRP) target triggered the amplification strategy. The Y-DNA probes (Y-DNA) were assembled using an assisted DNA probe (assisted DNA labeled with magnetic gold nanoparticles) which can hybridize to the captured DNA probe (consisting of aptamer and primer recognition region), and signal tags (NMOF encapsulating signal DNAs and different metal ions such as Pb(2+) or Cd(2+))...
May 22, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28377494/arabidopsis-fructokinase-like-protein-associations-are-regulated-by-atp
#19
John W Riggs, Judy Callis
The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated...
May 10, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28349355/polymerase-spiral-reaction-psr-a-novel-visual-isothermal-amplification-method-for-detection-of-canine-parvovirus-2-genomic-dna
#20
Vikas Gupta, Soumendu Chakravarti, Vishal Chander, Saurabh Majumder, Shabir Ahmad Bhat, Vivek Kumar Gupta, Sukdeb Nandi
Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples...
July 2017: Archives of Virology
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