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https://www.readbyqxmd.com/read/27907204/homologous-recombination-and-translesion-dna-synthesis-play-critical-roles-on-tolerating-dna-damage-caused-by-trace-levels-of-hexavalent-chromium
#1
Xu Tian, Keyur Patel, John R Ridpath, Youjun Chen, Yi-Hui Zhou, Dayna Neo, Jean Clement, Minoru Takata, Shunichi Takeda, Julian Sale, Fred A Wright, James A Swenberg, Jun Nakamura
Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19...
2016: PloS One
https://www.readbyqxmd.com/read/27907066/b-cell-based-seamless-engineering-of-antibody-fc-domains
#2
Koji Hashimoto, Kohei Kurosawa, Akiho Murayama, Hidetaka Seo, Kunihiro Ohta
Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody's Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the 'ADLib system')...
2016: PloS One
https://www.readbyqxmd.com/read/27832152/urocortin-treatment-improves-acute-hemodynamic-instability-and-reduces-myocardial-damage-in-post-cardiac-arrest-myocardial-dysfunction
#3
Chien-Hua Huang, Chih-Hung Wang, Min-Shan Tsai, Nai-Tan Hsu, Chih-Yen Chiang, Tzung-Dau Wang, Wei-Tien Chang, Huei-Wen Chen, Wen-Jone Chen
AIMS: Hemodynamic instability occurs following cardiac arrest and is associated with high mortality during the post-cardiac period. Urocortin is a novel peptide and a member of the corticotrophin-releasing factor family. Urocortin has the potential to improve acute cardiac dysfunction, as well as to reduce the myocardial damage sustained after ischemia reperfusion injury. The effects of urocortin in post-cardiac arrest myocardial dysfunction remain unclear. METHODS AND RESULTS: We developed a preclinical cardiac arrest model and investigated the effects of urocortin...
2016: PloS One
https://www.readbyqxmd.com/read/27815504/the-arrhythmogenic-calmodulin-mutation-d129g-dysregulates-cell-growth-calmodulin-dependent-kinase-ii-activity-and-cardiac-function-in-zebrafish
#4
Martin W Berchtold, Triantafyllos Zacharias, Katarzyna Kulej, Kevin Wang, Raffaela Torggler, Thomas Jespersen, Jau-Nian Chen, Martin R Larsen, Jonas M La Cour
Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets of which several are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability as well as heart rhythm remains unknown and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of wt CaM except for the LQTS mutant CaM D129G...
November 4, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27814490/mre11-is-essential-for-the-removal-of-lethal-topoisomerase-2-covalent-cleavage-complexes
#5
Nguyen Ngoc Hoa, Tsubasa Shimizu, Zhong Wei Zhou, Zhao-Qi Wang, Rajashree A Deshpande, Tanya T Paull, Salma Akter, Masataka Tsuda, Ryohei Furuta, Ken Tsusui, Shunichi Takeda, Hiroyuki Sasanuma
The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11(-/-)) and nuclease-deficient Mre11 (MRE11(-/H129N)) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons...
November 3, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27811920/acetylation-of-histone-h4-lysine-5-and-12-is-required-for-cenp-a-deposition-into-centromeres
#6
Wei-Hao Shang, Tetsuya Hori, Frederick G Westhorpe, Kristina M Godek, Atsushi Toyoda, Sadahiko Misu, Norikazu Monma, Kazuho Ikeo, Christopher W Carroll, Yasunari Takami, Asao Fujiyama, Hiroshi Kimura, Aaron F Straight, Tatsuo Fukagawa
Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A-H4-HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48-Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres...
November 4, 2016: Nature Communications
https://www.readbyqxmd.com/read/27701075/a-double-strand-break-can-trigger-immunoglobulin-gene-conversion
#7
Giulia Bastianello, Hiroshi Arakawa
All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease...
October 3, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27601656/in-b-cells-phosphatidylinositol-5-phosphate-4-kinase-%C3%AE-synthesizes-pi-4-5-p2-to-impact-mtorc2-and-akt-signaling
#8
Simon J Bulley, Alaa Droubi, Jonathan H Clarke, Karen E Anderson, Len R Stephens, Phillip T Hawkins, Robin F Irvine
Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P...
September 20, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27580264/determination-of-genotoxic-potential-by-comparison-of-structurally-related-azo-dyes-using-dna-repair-deficient-dt40-mutant-panels
#9
Masato Ooka, Koji Kobayashi, Takuya Abe, Kazuhiko Akiyama, Masahiko Hada, Shunichi Takeda, Kouji Hirota
Azo dyes, including Sudan I, Orange II and Orange G, are industrial dyes that are assumed to have genotoxic potential. However, neither the type of DNA damage induced nor the structural features responsible for toxicity have been determined. We used a panel of DNA-repair-pathway-deficient mutants generated from chicken DT40 cells to evaluate the ability of these azo dyes to induce DNA damage and to identify the type of DNA damage induced. We compared the structurally related azo dyes Sudan I, Orange II and Orange G to identify the structural features responsible for genotoxicity...
December 2016: Chemosphere
https://www.readbyqxmd.com/read/27530147/parp3-is-a-sensor-of-nicked-nucleosomes-and-monoribosylates-histone-h2b-glu2
#10
Gabrielle J Grundy, Luis M Polo, Zhihong Zeng, Stuart L Rulten, Nicolas C Hoch, Pathompong Paomephan, Yingqi Xu, Steve M Sweet, Alan W Thorne, Antony W Oliver, Steve J Matthews, Laurence H Pearl, Keith W Caldecott
PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate...
2016: Nature Communications
https://www.readbyqxmd.com/read/27524497/pold3-is-haploinsufficient-for-dna-replication-in-mice
#11
Matilde Murga, Emilio Lecona, Irene Kamileri, Marcos Díaz, Natalia Lugli, Sotirios K Sotiriou, Marta E Anton, Juan Méndez, Thanos D Halazonetis, Oscar Fernandez-Capetillo
The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals...
September 1, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27518625/determination-of-histone-h2ax-phosphorylation-in-dt40-cells
#12
Kana Nishihara, Sampada A Shahane, Menghang Xia
Visualization of DNA damage response protein recruitment to DNA damage sites enables measurement of the DNA damage. DNA double-strand breaks (DSBs) and blocked replication forks induce the phosphorylation of H2AX at serine 139 (γH2AX), and accumulate γH2AX which can then be detected as foci. The detection of γH2AX foci by immunostaining with antibodies that recognize γH2AX is an indicator of DSBs presence. This chapter describes the measurement of γH2AX immunostaining using a high-content imaging platform in chicken DT40 B-lymphocyte cell lines...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27515252/regulation-of-b-cell-differentiation-by-the-ubiquitin-binding-protein-tax1bp1
#13
Nobuko Matsushita, Midori Suzuki, Emi Ikebe, Shun Nagashima, Ryoko Inatome, Kenichi Asano, Masato Tanaka, Masayuki Matsushita, Eisaku Kondo, Hidekatsu Iha, Shigeru Yanagi
Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-κB (NF-κB) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27486975/poor-recognition-of-o6-isopropyl-dg-by-mgmt-triggers-double-strand-break-mediated-cell-death-and-micronucleus-induction-in-fanc-deficient-cells
#14
Kiyohiro Hashimoto, Vyom Sharma, Hiroyuki Sasanuma, Xu Tian, Minoru Takata, Shunichi Takeda, James A Swenberg, Jun Nakamura
Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS...
July 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27455635/shape-dependent-genotoxicity-of-mesoporous-silica-nanoparticles-and-cellular-mechanisms
#15
Meng Niu, Hongshan Zhong, Haibo Shao, Duo Hong, Tengchuang Ma, Ke Xu, Xiaowei Chen, Jinhang Han, Jun Sun
Although mesoporous silica nanoparticles (MSNs) are widely used in food products, cosmetics and nanomedicines as vector for drug delivery, data on their potential genotoxocity are limited. The aim of this study was to investigate the cytotoxic and genotoxic potentials of MSNs of different shapes, and to establish a high-throughput screening method for nanoparticles. We used functional macrophage receptor with collagenous structure (MARCO)-expressing DNA repair deficient chicken DT40 cells, which are designed to internalize nanoparticles and to be deficient in several specific DNA repair pathways...
March 2016: Journal of Nanoscience and Nanotechnology
https://www.readbyqxmd.com/read/27452521/loss-of-brca1-or-brca2-markedly-increases-the-rate-of-base-substitution-mutagenesis-and-has-distinct-effects-on-genomic-deletions
#16
J Zámborszky, B Szikriszt, J Z Gervai, O Pipek, Á Póti, M Krzystanek, D Ribli, J M Szalai-Gindl, I Csabai, Z Szallasi, C Swanton, A L Richardson, D Szüts
Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours...
July 25, 2016: Oncogene
https://www.readbyqxmd.com/read/27374870/positive-cofactor-4-pc4-is-critical-for-dna-repair-pathway-re-routing-in-dt40-cells
#17
Randolph B Caldwell, Herbert Braselmann, Ulrike Schoetz, Steffen Heuer, Harry Scherthan, Horst Zitzelsberger
PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27328625/roles-of-the-mitochondrial-na-ca-2-exchanger-nclx-in-b-lymphocyte-chemotaxis
#18
Bongju Kim, Ayako Takeuchi, Masaki Hikida, Satoshi Matsuoka
Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na(+)-dependent mitochondrial Ca(2+) efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca(2+) was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27311583/the-role-of-the-mre11-rad50-nbs1-complex-in-double-strand-break-repair-facts-and-myths
#19
Shunichi Takeda, Nguyen Ngoc Hoa, Hiroyuki Sasanuma
Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5'-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51-DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells...
August 2016: Journal of Radiation Research
https://www.readbyqxmd.com/read/27304067/characterization-of-staphylococcus-aureus-primosomal-dnad-protein-highly-conserved-c-terminal-region-is-crucial-for-ssdna-and-pria-helicase-binding-but-not-for-dnaa-protein-binding-and-self-tetramerization
#20
Yen-Hua Huang, Yi Lien, Chien-Chih Huang, Cheng-Yang Huang
The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD) and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204) showed a stable tetramer in solution...
2016: PloS One
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