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Polymerase substrate specificity

Yabin Liu, Erqiang Wei, Jian Zhao, Dexian Kong, Binghui Li
Silencing of tumor suppressor genes by hypermethylation in gene promoter regions is a crucial mechanism in carcinogenesis. Gene methylation may be reversible and evaluated easily, thus providing a promising substrate for the development of biomarkers for the detection and prevention of cancer, including colorectal cancer (CRC). In the present study, the protein expression and methylation status of smooth muscle protein 22α (SM22α) was investigated in 78 cases of CRC and adjacent normal tissue. The aim of the study was to investigate the function of SM22α in the pathogenesis of CRC and to identify a candidate biomarker for the early detection of CRC...
May 2018: Oncology Letters
Ling Liu, Xiaoyang Su, William J Quinn, Sheng Hui, Kristin Krukenberg, David W Frederick, Philip Redpath, Le Zhan, Karthikeyani Chellappa, Eileen White, Marie Migaud, Timothy J Mitchison, Joseph A Baur, Joshua D Rabinowitz
The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins...
April 20, 2018: Cell Metabolism
Paul L Boyer, Steven J Smith, Xue Zhi Zhao, Kalyan Das, Kevin Gruber, Eddy Arnold, Terrence R Burke, Stephen H Hughes
We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA/DNA heteroduplex. The compounds were tested using a variety of substrates: All three compounds inhibited the polymerase-independent RH activity of HIV-1 RT...
April 11, 2018: Journal of Virology
Si-Ying Cheng, Shu-Chao Wang, Ming Lei, Zhen Wang, Kun Xiong
Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly (ADP-ribose) polymerase 1-mediated parthanatos...
March 2018: Neural Regeneration Research
Thomas P Wytock, Aretha Fiebig, Jonathan W Willett, Julien Herrou, Aleksandra Fergin, Adilson E Motter, Sean Crosson
Cell growth is determined by substrate availability and the cell's metabolic capacity to assimilate substrates into building blocks. Metabolic genes that determine growth rate may interact synergistically or antagonistically, and can accelerate or slow growth, depending on genetic background and environmental conditions. We evolved a diverse set of Escherichia coli single-gene deletion mutants with a spectrum of growth rates and identified mutations that generally increase growth rate. Despite the metabolic differences between parent strains, mutations that enhanced growth largely mapped to core transcription machinery, including the β and β' subunits of RNA polymerase (RNAP) and the transcription elongation factor, NusA...
March 27, 2018: PLoS Genetics
Shijun Yu, Li Li, Qiong Wu, Ning Dou, Yandong Li, Yong Gao
Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase that regulates the basic activities of eukaryotes by dephosphorylating its substrates. The function and substrate specificity of PP2A are generally determined by its regulatory subunits. In the present study, the clinical significance and roles of PPP2R2D, one of the regulatory subunits of PP2A, were demonstrated in gastric cancer (GC) carcinogenesis. Through a tissue microarray and quantitative polymerase chain reaction analysis, it was demonstrated that PPP2R2D was commonly upregulated in GC samples...
March 20, 2018: International Journal of Oncology
Shibao Wang, Yinghui Huang, Xupeng Mu, Tianyang Qi, Sha Qiao, Zhenxia Lu, Hongjun Li
The present study aimed to explore the relationship between DNA methylation and breast cancer under different cell culture conditions. MCF‑7 breast cancer cells were cultured in two‑dimensional (2D), three‑dimensional (3D) and orthotopic transplantation (Ti) adhesion substrates. Principal component analysis (PCA) was used for global visualization of these three samples. The methylation status of CpG sites was examined by unsupervised clustering analysis. Scatter plots and histograms were constructed from the mean β‑values from 3D vs...
March 14, 2018: Molecular Medicine Reports
Lisa N Chesner, Colin Campbell
The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology...
March 5, 2018: Journal of Visualized Experiments: JoVE
Alex Pines, Madelon Dijk, Matthew Makowski, Elisabeth M Meulenbroek, Mischa G Vrouwe, Yana van der Weegen, Marijke Baltissen, Pim J French, Martin E van Royen, Martijn S Luijsterburg, Leon H Mullenders, Michiel Vermeulen, Wim Vermeulen, Navraj S Pannu, Haico van Attikum
Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA ). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin...
March 12, 2018: Nature Communications
E A Belousova, А A Ishchenko, O I Lavrik
Most members of the poly(ADP-ribose)polymerase family, PARP family, have a catalytic activity that involves the transfer of ADP-ribose from a beta-NAD+-molecule to protein acceptors. It was recently discovered by Talhaoui et al. that DNA-dependent PARP1 and PARP2 can also modify DNA. Here, we demonstrate that DNA-dependent PARP3 can modify DNA and form a specific primed structure for further use by the repair proteins. We demonstrated that gapped DNA that was ADP-ribosylated by PARP3 could be ligated to double-stranded DNA by DNA ligases...
March 8, 2018: Scientific Reports
Soo-Yeon Kim, Seul-Ki Kwon, So-Young Lee, Kwang-Hyun Baek
Most proteins undergo ubiquitination, a process by which ubiquitin proteins bind to their substrate proteins; by contrast, deubiquitination is a process that reverses ubiquitination. Deubiquitinating enzymes (DUBs) function to remove ubiquitin proteins from the protein targets and serve an essential role in regulating DNA repair, protein degradation, apoptosis and immune responses. Abnormal regulation of DUBs may affect a number of cellular processes and may lead to a variety of human diseases, including cancer...
March 5, 2018: International Journal of Oncology
Jessica Cusato, Amedeo De Nicolò, Lucio Boglione, Fabio Favata, Alessandra Ariaudo, Simone Mornese Pinna, Chiara Carcieri, Federica Guido, Valeria Avataneo, Giuseppe Cariti, Giovanni Di Perri, Antonio D'Avolio
Background: Sofosbuvir is a potent nucleotide HCV NS5B polymerase inhibitor that is also a P-glycoprotein (encoded by the ABCB1 gene) and breast cancer resistance protein (encoded by the ABCG2 gene) substrate. Concerning previous anti-HCV therapies, pharmacogenetics had a significant impact, particularly considering the association of interleukin28B polymorphisms with dual-therapy (ribavirin + pegylated IFN) outcomes. Objectives: In this work, we investigated the association between sofosbuvir and its prevalent metabolite (GS-331007) plasma concentrations at 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCG2 and HNF4α) related to sofosbuvir transport...
March 2, 2018: Journal of Antimicrobial Chemotherapy
Manuela Gast, Stefanie Kühner, Harald Sobek, Paul Walther, Boris Mizaikoff
Inspired by the recognition processes found in biology such as enzyme-substrate and antibody-antigen interactions, synthetic systems with comparable molecular recognition properties have been investigated during recent years based on molecular imprinting strategies. While materials with recognition capabilities for small molecules (i.e., with low molecular weight) have achieved substantial advancements, the synthesis of molecularly imprinted materials with virus recognition properties remains challenging to date...
May 1, 2018: Analytical Chemistry
Kai-Chih Chang, Chin-Yi Chung, Chen-Hsing Yeh, Kuo-Hsiu Hsu, Ya-Ching Chin, Sin-Siang Huang, Bo-Rong Liu, Hsi-An Chen, Anren Hu, Po-Chi Soo, Wen-Ping Peng
The appearance and spread of carbapenem-resistant Acinetobacter baumannii (CRAB) pose a challenge for optimization of antibiotic therapies and outbreak preventions. The carbapenemase production can be detected through culture-based methods (e.g. Modified Hodge Test-MHT) and DNA based methods (e.g. Polymerase Chain Reaction-PCR). The culture-based methods are time-consuming, whereas those of PCR assays need only a few hours but due to its specificity, can only detect known genetic targets encoding carbapenem-resistance genes...
April 2018: Journal of Microbiological Methods
Courtney R Sullivan, Rachael H Koene, Kathryn Hasselfeld, Sinead M O'Donovan, Amy Ramsey, Robert E McCullumsmith
Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays...
March 1, 2018: Molecular Psychiatry
Haolin Liu, Chao Wang, Schuyler Lee, Fangkun Ning, Yang Wang, Qianqian Zhang, Zhongzhou Chen, Jianye Zang, Jay Nix, Shaodong Dai, Philippa Marrack, James Hagman, John Kappler, Gongyi Zhang
We have reported that JMJD5 and JMJD7 (JMJD5/7) are responsible for the clipping of arginine methylated histone tails to generate "tailless nucleosomes", which could release the pausing RNA polymerase II (Pol II) into productive transcription elongation. JMJD5/7 function as endopeptidases that cleave histone tails specifically adjacent to methylated arginine residues and continue to degrade N-terminal residues of histones via their aminopeptidase activity. Here, we report structural and biochemical studies on JMJD5/7 to understand the basis of substrate recognition and catalysis mechanism by this JmjC subfamily...
February 19, 2018: Scientific Reports
Fiyinfoluwa A Adesioye, Thulani P Makhalanyane, Surendra Vikram, Bryan T Sewell, Wolf-Dieter Schubert, Don A Cowan
A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1 ). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1 , respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2...
April 15, 2018: Applied and Environmental Microbiology
Virginia Neyro, Valéry Elie, Yves Médard, Evelyne Jacqz-Aigrain
Growth and maturation changes are mainly responsible for differences in drug pharmacokinetics and pharmacodynamics observed between adults and children, especially neonates. Ontogeny of drug-metabolizing enzymes and transporters plays an important role in drugs interindividual pharmacokinetic variability but data are limited in both term and preterm neonates. This study aimed to characterize mRNA expression of the main drug-metabolizing enzymes and transport proteins involved in drug disposition, using umbilical cord blood (UCB), according to gender, gestational age, and genetic background...
February 13, 2018: Fundamental & Clinical Pharmacology
Vito Genna, Paolo Carloni, Marco De Vivo
Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is critical for DNA and RNA functioning. Yet, the molecular basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large data set of Pol/DNA structures, we first describe the conserved presence of one positively charged residue (Lys or Arg), which is similarly located near the enzymatic two-metal active site, always interacting directly with the incoming substrate (d)NTP...
March 7, 2018: Journal of the American Chemical Society
Manal S Zaher, Fahad Rashid, Bo Song, Luay I Joudeh, Mohamed A Sobhy, Muhammad Tehseen, Manju M Hingorani, Samir M Hamdan
RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively...
April 6, 2018: Nucleic Acids Research
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