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Polymerase substrate specificity

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 Kuehner, 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 remain challenging to date...
March 5, 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...
February 27, 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 dataset was screened in-silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded a ∼36 kDa protein (Axe1 NaM1 ). The synthesised gene was cloned, expressed and the resulting protein, purified. NaM1 was optimally active at pH 8.5 and 30°C, and functionally stable at salt concentrations up to 5 M. The specific activity and catalytic efficiency were 488.9 Umg -1 and 3.26x10 6 M -1 s -1 , respectively. The crystal structure of wild type NaM1 was solved at a resolution of 2...
February 16, 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 play an important role in drugs inter-individual 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 dataset 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...
February 9, 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...
February 6, 2018: Nucleic Acids Research
Wen Jing Zhu, Masato Kobayashi, Kohei Yamada, Kodai Nishi, Kotaro Takahashi, Asuka Mizutani, Ryuichi Nishii, Leo G Flores, Naoto Shikano, Munetaka Kunishima, Keiichi Kawai
INTRODUCTION: Due to its poor prognosis, specific imaging for early detection of malignant melanoma is strongly desired. Although radioiodine-labeled 4-hydroxyphenylcysteamine, which we previously developed, has good affinity for tyrosinase, an enzyme in the melanin metabolic pathway, image contrast of the melanoma:organ ratios is not sufficiently high for detection of primary melanoma and metastases at early injection times. In this study, we developed radioiodine-labeled acetaminophen (I-AP) for specific, high-contrast imaging of malignant melanoma...
January 3, 2018: Nuclear Medicine and Biology
Huijuan Cheng, Weiwei Wang, Guangke Wang, Anran Wang, Linfang Du, Weihua Lou
BACKGROUND Ras-related C3 botulinum toxin substrate 1 (Rac1) is implicated in a variety of cellular functions and is related to tumor growth and metastasis. This study aimed to explore the role of Rac1 in hypopharyngeal squamous cell carcinoma (HSCC). MATERIAL AND METHODS The Rac1 expression in HSCC tissues was determined by quantitative real-time polymerase chain reaction and Western blot analysis. The level of Rac1 in HSCC cells was downregulated by a Rac1-specific shRNA. Then, the growth and metastasis of HSCC cells were assessed in vitro by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry, Hoechst staining, and Transwell assay...
February 7, 2018: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
Nathan H Blewett, Richard J Maraia
The conserved nuclear RNA-binding factor known as La protein arose in an ancient eukaryote, phylogenetically associated with another eukaryotic hallmark, synthesis of tRNA by RNA polymerase III (RNAP III). Because 3'-oligo(U) is the sequence-specific signal for transcription termination by RNAP III as well as the high affinity binding site for La, the latter is linked to the intranuclear posttranscriptional processing of eukaryotic precursor-tRNAs. The pre-tRNA processing pathway must accommodate a variety of substrates that are destined for both common steps as well as tRNA-specific events...
January 31, 2018: Biochimica et Biophysica Acta
Xianfang Jiang, Yanping Zhong, Li Zheng, Jinmin Zhao
Autologous chondrocyte implantation (ACI) has emerged as a novel approach to cartilage repair through the use of harvested chondrocytes. However, the expansion of the chondrocytes from the donor tissue in vitro is restricted by the limited cell numbers and the dedifferentiation of the chondrocytes. The present study investigated the effect of collagen-based films, including collagen, hydroxyapatite (HA)/collagen (HC) and in situ synthesis of nano‑HC (nHC), on monolayer cultures of chondrocytes. As a substrate for the chondrocytes monolayer culture in vitro, nHC was able to restrain the dedifferentiation of chondrocytes and facilitate cell expansion, which was detected by methyl thiazolyl tetrazolium assay, scanning electron microscopy, calcein‑acetoxymethyl/propidium iodide staining, hematoxylin and eosin staining, Safranin O staining, immunohistochemical staining and reverse transcription‑quantitative polymerase chain reaction...
January 26, 2018: International Journal of Molecular Medicine
Gabriella Zarkovic, Ekaterina A Belousova, Ibtissam Talhaoui, Christine Saint-Pierre, Mikhail M Kutuzov, Bakhyt T Matkarimov, Denis Biard, Didier Gasparutto, Olga I Lavrik, Alexander A Ishchenko
Poly(ADP-ribose) polymerases (PARPs) act as DNA break sensors and catalyze the synthesis of polymers of ADP-ribose (PAR) covalently attached to acceptor proteins at DNA damage sites. It has been demonstrated that both mammalian PARP1 and PARP2 PARylate double-strand break termini in DNA oligonucleotide duplexes in vitro. Here, we show that mammalian PARP2 and PARP3 can PARylate and mono(ADP-ribosyl)ate (MARylate), respectively, 5'- and 3'-terminal phosphate residues at double- and single-strand break termini of a DNA molecule containing multiple strand breaks...
January 18, 2018: Nucleic Acids Research
Alicia D D'Souza, Boris P Belotserkovskii, Philip C Hanawalt
The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e...
January 18, 2018: Biochimica et Biophysica Acta
Mikhail Sergeevich Zastrozhin, Elena Anatolievna Grishina, Kristina Anatolievna Ryzhikova, Valery Valerievich Smirnov, Ludmila Mikhailovna Savchenko, Evgeny Alekseevich Bryun, Dmitry Alekseevich Sychev
Background: Isoenzymes CYP2D6 and CYP3A4, the activity of which varies widely, are involved in metabolism of haloperidol and may influence its profile of efficacy and safety. Objective: The primary aim of this study was to estimate the relationship between CYP3A5 gene polymorphism, activity of the CYP3A isoenzyme, and the risk of development of adverse drug reactions by haloperidol in patients with alcohol abuse. Methods: Sixty-six male alcohol-addicted patients participated in the study...
2018: Pharmacogenomics and Personalized Medicine
Shaohua Zhang, Xuepeng Cai, Xuenong Luo, Shuai Wang, Aijiang Guo, Junling Hou, Run Wu
Leucine aminopeptidase (LAP, EC: is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa...
January 9, 2018: Experimental Parasitology
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