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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
#1
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/28617439/cdc20-directs-proteasome-mediated-degradation-of-the-tumor-suppressor-smar1-in-higher-grades-of-cancer-through-the-anaphase-promoting-complex
#2
Debasish Paul, Suvankar Ghorai, U S Dinesh, Praveenkumar Shetty, Samit Chattopadhyay, Manas Kumar Santra
The Tumor suppressor SMAR1 (scaffold matrix attachment region binding protein 1) has a crucial role in maintaining genomic stability, cell cycle progression and apoptosis.Our previous finding showed that it is highly suppressed in higher grade of cancer. However, the underlying mechanism of this suppression was not well understood. In this study, we show that SMAR1 expression levels are controlled at the proteasomal level by five RING finger E3 ubiquitin ligases including, Cdc20, a substrate receptor of ubiquitin ligase APC/C complex...
June 15, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28611047/smurf2-mediated-stabilization-of-dna-topoisomerase-ii%C3%AE-controls-genomic-integrity
#3
Andrea Emanuelli, Aurora P Borroni, Liat Apel-Sarid, Pooja A Shah, Dhanoop Manikoth Ayyathan, Praveen Koganti, Gal Levy-Cohen, Michael Blank
DNA topoisomerase IIα (Topo IIα) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo IIα function is well understood, but how its expression is regulated remains unclear. Here we identify the E3 ubiquitin ligase Smurf2 as a physiological regulator of Topo IIα levels. Smurf2 physically interacted with Topo IIα and modified its ubiquitination status to protect Topo IIα from the proteasomal degradation in dose- and catalytically-dependent manners...
June 13, 2017: Cancer Research
https://www.readbyqxmd.com/read/28604743/shortage-of-dntps-underlies-altered-replication-dynamics-and-dna-breakage-in-the-absence-of-the-apc-c-cofactor-cdh1
#4
J Garzón, R Rodríguez, Z Kong, A Chabes, S Rodríguez-Acebes, J Méndez, S Moreno, I García-Higuera
The APC/C-Cdh1 ubiquitin-ligase complex targets cell cycle regulators for proteosomal degradation and helps prevent tumor development and accumulation of chromosomal aberrations. Replication stress has been proposed to be the main driver of genomic instability in the absence of Cdh1, but the real contribution of APC/C-Cdh1 to efficient replication, especially in normal cells, remains unclear. Here we show that, in primary MEFs, acute depletion or permanent ablation of Cdh1 slowed down replication fork movement and increased origin activity...
June 12, 2017: Oncogene
https://www.readbyqxmd.com/read/28594266/simple-fluorescence-based-detection-of-protein-kinase-a-activity-using-a-molecular-beacon-probe
#5
Changbei Ma, Xiaoyuan Lv, Kemin Wang, Shunxin Jin, Haisheng Liu, Kefeng Wu, Weimin Zeng
Protein kinase A was detected by quantifying the amount of ATP utilized after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A...
June 8, 2017: Bioengineered
https://www.readbyqxmd.com/read/28590593/the-proteasome-enters-the-meiotic-prophase-fray
#6
REVIEW
Aleksandar Vujin, Monique Zetka
The segregation of homologous chromosomes in meiosis depends on their ability to locate one another in the nucleus and establish a physical association through crossing over. A tightly regulated number of crossovers (COs) emerges following repair of induced DNA double-strand breaks by homologous recombination (HR), but the process of how HR intermediates transition into COs is still poorly understood. Two recent studies by Ahuja et al. and Rao et al. have revealed a role for chromosomally localized proteasomes in choreographing both homologous chromosome pairing and the evolution of HR intermediates into segregation-competent COs...
June 7, 2017: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
https://www.readbyqxmd.com/read/28587922/molecular-basis-for-k63-linked-ubiquitination-processes-in-double-strand-dna-break-repair-a-focus-on-kinetics-and-dynamics
#7
REVIEW
Brian L Lee, Anamika, J N Mark Glover, Michael J Hendzel, Leo Spyracopoulos
Cells are exposed to thousands of DNA damage events on a daily basis. This damage must be repaired to preserve genetic information, and prevent development of disease. The most deleterious damage is a double strand break (DSB), which is detected and repaired by mechanisms known as non-homologous end joining (NHEJ), and homologous recombination (HR), components of the DNA damage response system. NHEJ is an error prone first line of defense, whereas HR invokes error free repair, and is the focus of this review...
June 3, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28582638/association-of-cd147-genetic-polymorphisms-with-carotid-atherosclerotic-plaques-in-a-han-chinese-population-with-cerebral-infarction
#8
Tongtian Ni, Min Chen, Kang Yang, Jianwei Shao, Yi Fu, Weijun Zhou
INTRODUCTION: Given the important role of CD147 in the development of atherosclerosis, we speculated that CD147 genetic polymorphisms might influence the formation of carotid atherosclerotic plaques. The study was to investigate the association between CD147 gene polymorphisms and susceptibility to carotid atherosclerotic plaques in individuals with cerebral infarction (CI). METHODS: Eight SNPs in the regulatory and coding regions of the CD147 gene were examined using polymerase chain reaction-ligase detection reaction (PCR-LDR) in DNA samples from 732 Chinese patients with CI, divided into a carotid plaque group (n=475) and a non-carotid plaque group (n=257)...
May 26, 2017: Thrombosis Research
https://www.readbyqxmd.com/read/28579617/hdac1-2-inhibition-and-doxorubicin-impair-mre11-dependent-dna-repair-and-disc-to-override-bcr-abl1-driven-dsb-repair-in-philadelphia-chromosome-positive-b-cell-precursor-acute-lymphoblastic-leukemia
#9
S T Promod, D P Johnson, S E Bennett, E M Dennis, B G Banowsky, S S Jones, J R Shearstone, S N Quayle, C Min, M Jarpe, T Mosbruger, A D Pomicter, R R Miles, W Y Chen, K N Bhalla, P A Zweidler-McKay, D C Shrieve, M W Deininger, M B Chandrasekharan, S Bhaskara
Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here, we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms...
June 5, 2017: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/28577191/deletion-analysis-of-gh7-endoglucanase-gene-cel7b-promoter-region-in-a-talaromyces-cellulolyticus-ligd-disrupted-strain
#10
Tatsuya Fujii, Hiroyuki Inoue, Kazuhiko Ishikawa, Tamotsu Hoshino
Talaromyces cellulolyticus is expected to become an industrial cellulase producer. In this study, we performed deletion analysis of the promoter region of the GH7 endoglucanase gene (cel7B), which encodes one of the major cellulases, using a β-glucuronidase reporter system. To obtain strains that harbor each gene cassette at the same locus, we had to improve the homologous recombination frequency. Hence, the ligD gene, encoding DNA ligase IV, was disrupted by homologous recombination. After that, the introduced pyrF marker gene, encoding orotate phosphoribosyl transferase, was deleted by a marker recycling system...
June 2, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28575658/rfwd3-mediated-ubiquitination-promotes-timely-removal-of-both-rpa-and-rad51-from-dna-damage-sites-to-facilitate-homologous-recombination
#11
Shojiro Inano, Koichi Sato, Yoko Katsuki, Wataru Kobayashi, Hiroki Tanaka, Kazuhiro Nakajima, Shinichiro Nakada, Hiroyuki Miyoshi, Kerstin Knies, Akifumi Takaori-Kondo, Detlev Schindler, Masamichi Ishiai, Hitoshi Kurumizaka, Minoru Takata
RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation...
June 1, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28575657/rpa-mediated-recruitment-of-the-e3-ligase-rfwd3-is-vital-for-interstrand-crosslink-repair-and-human-health
#12
Laura Feeney, Ivan M Muñoz, Christophe Lachaud, Rachel Toth, Paul L Appleton, Detlev Schindler, John Rouse
Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling...
June 1, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28573495/rnf43-is-mutated-less-frequently-in-lynch-syndrome-compared-with-sporadic-microsatellite-unstable-colorectal-cancers
#13
Lochlan J Fennell, Mark Clendenning, Diane M McKeone, Saara H Jamieson, Samanthy Balachandran, Jennifer Borowsky, John Liu, Futoshi Kawamata, Catherine E Bond, Christophe Rosty, Matthew E Burge, Daniel D Buchanan, Barbara A Leggett, Vicki L J Whitehall
The WNT signaling pathway is commonly altered during colorectal cancer development. The E3 ubiquitin ligase, RNF43, negatively regulates the WNT signal through increased ubiquitination and subsequent degradation of the Frizzled receptor. RNF43 has recently been reported to harbor frequent truncating frameshift mutations in sporadic microsatellite unstable (MSI) colorectal cancers. This study assesses the relative frequency of RNF43 mutations in hereditary colorectal cancers arising in the setting of Lynch syndrome...
June 1, 2017: Familial Cancer
https://www.readbyqxmd.com/read/28573017/a-crispr-cas9-high-throughput-genome-editing-toolkit-for-kinetoplastids
#14
Tom Beneke, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, Eva Gluenz
Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit...
May 2017: Royal Society Open Science
https://www.readbyqxmd.com/read/28562614/properties-of-genes-essential-for-mouse-development
#15
Mitra Kabir, Ana Barradas, George T Tzotzos, Kathryn E Hentges, Andrew J Doig
Essential genes are those that are critical for life. In the specific case of the mouse, they are the set of genes whose deletion means that a mouse is unable to survive after birth. As such, they are the key minimal set of genes needed for all the steps of development to produce an organism capable of life ex utero. We explored a wide range of sequence and functional features to characterise essential (lethal) and non-essential (viable) genes in mice. Experimental data curated manually identified 1301 essential genes and 3451 viable genes...
2017: PloS One
https://www.readbyqxmd.com/read/28552615/sumo-targeted-dna-translocase-rrp2-protects-the-genome-from-top2-induced-dna-damage
#16
Yi Wei, Li-Xue Diao, Shan Lu, Hai-Tao Wang, Fang Suo, Meng-Qiu Dong, Li-Lin Du
The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons. Loss of Rrp2 enhances SUMOylation-dependent ubiquitination and degradation of Top2, which in turn increases DNA damage at sites where Top2-DNA complexes are trapped...
June 1, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28550017/a-lysine-desert-protects-a-novel-domain-in-the-slx5-slx8-sumo-targeted-ub-ligase-to-maintain-sumoylation-levels-in-saccharomyces-cerevisiae
#17
Pragati Sharma, Janet R Mullen, Minxing Li, Mikel Zaratiegui, Samuel F Bunting, Steven J Brill
Protein modification by the small ubiquitin-like modifier (SUMO) plays important roles in genome maintenance. In Saccharomyces cerevisiae, proper regulation of sumoylation is known to be essential for viability in certain DNA repair mutants. Here we find the opposite result; proper regulation of sumoylation is lethal in certain DNA repair mutants. Yeast cells lacking the repair factors TDP1 and WSS1 are synthetically lethal due to their redundant roles in removing Top1-DNA covalent complexes (Top1ccs). A screen for suppressors of tdp1∆ wss1∆ synthetic lethality isolated mutations in genes known to control global sumoylation levels including ULP1, ULP2, SIZ2 and SLX5 The results suggest that alternative pathways of repair become available when sumoylation levels are altered...
May 26, 2017: Genetics
https://www.readbyqxmd.com/read/28542143/dna-damage-and-s-phase-dependent-e2f1-stabilization-requires-the-ciap1-e3-ubiquitin-ligase-and-is-associated-with-k63-poly-ubiquitination-on-lysine-161-164-residues
#18
Valérie Glorian, Jennifer Allègre, Jean Berthelet, Baptiste Dumetier, Pierre-Marie Boutanquoi, Nathalie Droin, Cémile Kayaci, Jessy Cartier, Simon Gemble, Guillaume Marcion, Daniel Gonzalez, Romain Boidot, Carmen Garrido, Olivier Michaud, Eric Solary, Laurence Dubrez
The E2F transcription factor 1 is subtly regulated along the cell cycle progression and in response to DNA damage by post-translational modifications. Here, we demonstrated that the E3-ubiquitin ligase cellular inhibitor of apoptosis 1 (cIAP1) increases E2F1 K63-poly-ubiquitination on the lysine residue 161/164 cluster, which is associated with the transcriptional factor stability and activity. Mutation of these lysine residues completely abrogates the binding of E2F1 to CCNE, TP73 and APAF1 promoters, thus inhibiting transcriptional activation of these genes and E2F1-mediated cell proliferation control...
May 25, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28540703/a-novel-platform-for-high-throughput-gene-synthesis-to-maximize-recombinant-expression-in-escherichia-coli
#19
Ana Filipa Sequeira, Joana L A Brás, Vânia O Fernandes, Catarina I P D Guerreiro, Renaud Vincentelli, Carlos M G A Fontes
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28540698/introduction-on-using-the-fastpcr-software-and-the-related-java-web-tools-for-pcr-and-oligonucleotide-assembly-and-analysis
#20
Ruslan Kalendar, Timofey V Tselykh, Bekbolat Khassenov, Erlan M Ramanculov
This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP)...
2017: Methods in Molecular Biology
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