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Uracil dna glycosylase

Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, Xing Chang
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants...
October 10, 2016: Nature Methods
Javier Abellón-Ruiz, Sonoko Ishino, Yoshizumi Ishino, Bernard A Connolly
In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine...
2016: Archaea: An International Microbiological Journal
Mélanie Flaender, Guillaume Costa, Guillaume Nonglaton, Christine Saint-Pierre, Didier Gasparutto
DNA is under continuous assault by environmental and endogenous reactive oxygen and alkylating species, inducing the formation of mutagenic, toxic and genome destabilizing nucleobase lesions. Due to the implications of such genetic alterations in cell death, aging, inflammation, neurodegenerative diseases and cancer, many efforts have been devoted to developing assays that aim at analyzing DNA repair activities from purified enzymes or cell extracts. The present work deals with the conception and application of a new, miniaturized and parallelized on surface-DNA biosensor to measure base excision repair (BER) activities...
September 22, 2016: Analyst
Elena M Cortizas, Astrid Zahn, Shiva Safavi, Joseph A Reed, Francisco Vega, Javier M Di Noia, Ramiro E Verdun
Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG)...
October 3, 2016: Journal of Experimental Medicine
Norbert Schormann, Natalia Zhukovskaya, Gregory Bedwell, Manunya Nuth, Richard Gillilan, Peter E Prevelige, Robert P Ricciardi, Surajit Banerjee, Debasish Chattopadhyay
Uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication...
September 29, 2016: Protein Science: a Publication of the Protein Society
Naile Dame-Teixeira, Clarissa Cavalcanti Fatturi Parolo, Marisa Maltz, Aradhna Tugnait, Deirdre Devine, Thuy Do
BACKGROUND: The studies of the distribution of Actinomyces spp. on carious and non-carious root surfaces have not been able to confirm the association of these bacteria with root caries, although they were extensively implicated as a prime suspect in root caries. OBJECTIVE: The aim of this study was to observe the gene expression of Actinomyces spp. in the microbiota of root surfaces with and without caries. DESIGN: The oral biofilms from exposed sound root surface (SRS; n=10) and active root caries (RC; n=30) samples were collected...
2016: Journal of Oral Microbiology
Céline Contesto-Richefeu, Nicolas Tarbouriech, Xavier Brazzolotto, Wim P Burmeister, Christophe N Peyrefitte, Frédéric Iseni
The Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation...
September 2016: Acta Crystallographica. Section F, Structural Biology Communications
Changbei Ma, Kefeng Wu, Haisheng Liu, Kun Xia, Kemin Wang, Jun Wang
We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0...
November 1, 2016: Talanta
Christopher T Coey, Shuja S Malik, Lakshmi S Pidugu, Kristen M Varney, Edwin Pozharski, Alexander C Drohat
Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG(82-308), a new construct containing 29 more N-terminal residues than TDG(111-308), the construct used for previous structures of DNA-bound TDG...
August 31, 2016: Nucleic Acids Research
Shannen L Cravens, James T Stivers
The energetic nature of the interactions of DNA base excision repair glycosylases with undamaged and damaged DNA and the nuclear environment are expected to significantly impact the time it takes for these enzymes to search for damaged DNA bases. In particular, the high concentration of monovalent ions, macromolecule crowding, and densely packed DNA chains in the cell nucleus could alter the search mechanisms of these enzymes as compared to findings in dilute buffers typically used in in vitro experiments. Here we utilize an in vitro system where the concerted effects of monovalent ions, macromolecular crowding, and high concentrations of bulk DNA chains on the activity of two paradigm human DNA glycosylases can be determined...
September 20, 2016: Biochemistry
Ying Wu, Xiaohong Zhou, Christopher O Barnes, Maria DeLucia, Aina E Cohen, Angela M Gronenborn, Jinwoo Ahn, Guillermo Calero
The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4-RING E3 ubiquitin ligase (CRL4) of the host ubiquitin-proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1-DCAF1-HIV-1-Vpr-uracil-DNA glycosylase (UNG2) complex...
October 2016: Nature Structural & Molecular Biology
Yan Yan, Xiangzi Han, Yulan Qing, Allison G Condie, Shashank Gorityala, Shuming Yang, Yan Xu, Youwei Zhang, Stanton L Gerson
5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines...
August 9, 2016: Oncotarget
Ki Soo Park, Chang Yeol Lee, Kyoung Suk Kang, Hyun Gyu Park
We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target-triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe...
July 12, 2016: Biosensors & Bioelectronics
Keiji Nishida, Takayuki Arazoe, Nozomu Yachie, Satomi Banno, Mika Kakimoto, Mayura Tabata, Masao Mochizuki, Aya Miyabe, Michihiro Araki, Kiyotaka Y Hara, Zenpei Shimatani, Akihiko Kondo
The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis...
September 16, 2016: Science
Yi-Chen Du, Li-Na Zhu, De-Ming Kong
To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms...
December 15, 2016: Biosensors & Bioelectronics
John Lalith Charles Richard, Manu Shubhdarshan Shukla, Hervé Menoni, Khalid Ouararhni, Imtiaz Nisar Lone, Yohan Roulland, Christophe Papin, Elsa Ben Simon, Tapas Kundu, Ali Hamiche, Dimitar Angelov, Stefan Dimitrov
FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA...
July 2016: PLoS Genetics
Kasia Hrecka, Caili Hao, Ming-Chieh Shun, Sarabpreet Kaur, Selene K Swanson, Laurence Florens, Michael P Washburn, Jacek Skowronski
HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized...
July 5, 2016: Proceedings of the National Academy of Sciences of the United States of America
Yi Tang, Hao Chen, Youxiang Diao
Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 10(0.89) - 1 × 10(1...
June 7, 2016: Scientific Reports
Sibel Kucukyildirim, Hongan Long, Way Sung, Samuel F Miller, Thomas G Doak, Michael Lynch
Mycobacterium smegmatis is a bacterium that is naturally devoid of known postreplicative DNA mismatch repair (MMR) homologs, mutS and mutL, providing an opportunity to investigate how the mutation rate and spectrum has evolved in the absence of a highly conserved primary repair pathway. Mutation accumulation experiments of M. smegmatis yielded a base-substitution mutation rate of 5.27 × 10(-10) per site per generation, or 0.0036 per genome per generation, which is surprisingly similar to the mutation rate in MMR-functional unicellular organisms...
2016: G3: Genes—Genomes—Genetics
Natalya A Torgasheva, Natalya I Menzorova, Yurii T Sibirtsev, Valery A Rasskazov, Dmitry O Zharkov, Georgy A Nevinsky
In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis...
June 21, 2016: Molecular BioSystems
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