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https://www.readbyqxmd.com/read/29027452/-expression-and-activity-assay-of-recombinant-flap-endonuclease-1
#1
Nan Sheng, Yinjiao Ma, Jianping Wang, Bingjie Zou, Guohua Zhou
Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28974572/whole-genome-sequence-analysis-of-mutations-accumulated-in-rad27%C3%AE-%C3%A2-yeast-strains-with-defects-in-the-processing-of-okazaki-fragments-indicates-template-switching-events
#2
Sumita Omer, Bar Lavi, Piotr A Mieczkowski, Shay Covo, Einat Hazkani-Covo
Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a Saccharomyces cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing...
October 3, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28973450/complementation-of-aprataxin-deficiency-by-base-excision-repair-enzymes-in-mitochondrial-extracts
#3
Melike Çaglayan, Rajendra Prasad, Rachel Krasich, Matthew J Longley, Kei Kadoda, Masataka Tsuda, Hiroyuki Sasanuma, Shunichi Takeda, Keizo Tano, William C Copeland, Samuel H Wilson
Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5'-adenylate (5'-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5'-AMP or 5'-adenylated-deoxyribose phosphate (5'-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency...
September 29, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28912877/fen1-knockdown-improves-trastuzumab-sensitivity-in-human-epidermal-growth-factor-2-positive-breast-cancer-cells
#4
Xue Zeng, Xiaofang Che, Yun-Peng Liu, Xiu-Juan Qu, Lu Xu, Chen-Yang Zhao, Chun-Lei Zheng, Ke-Zuo Hou, Yuee Teng
Trastuzumab has been widely applied as a treatment for human epidermal growth factor 2 (HER2)-overexpressing breast cancer. However, the therapeutic efficacy of trastuzumab is limited. Flap endonuclease 1 (FEN1) is a multifunctional endonuclease that has a crucial role in DNA recombination and repair. Inhibition of FEN1 is associated with the reversal of anticancer drug resistance. However, it is unclear whether FEN1 is involved in trastuzumab resistance. In the present study, it was demonstrated that trastuzumab increases the expression of FEN1, and FEN1 knockdown significantly enhanced the sensitivity of BT474 cells to trastuzumab (P<0...
October 2017: Experimental and Therapeutic Medicine
https://www.readbyqxmd.com/read/28893156/erythrosine-b-and-quinoline-yellow-dyes-regulate-dna-repair-gene-expression-in-human-hepg2-cells
#5
Farah Md Chequer, Vinicius P Venancio, Mara R Almeida, Alexandre F Aissa, Maria Lourdes P Bianchi, Lusânia Mg Antunes
Erythrosine B (ErB) is a cherry pink food colorant and is widely used in foods, drugs, and cosmetics. Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application to the skin, lips, and/or body surface. Previously, ErB and QY synthetic dyes were found to induce DNA damage in HepG2 cells. The aim of this study was to investigate the molecular basis underlying the genotoxicity attributed to ErB and QY using the RT(2) Profiler polymerase chain reaction array and by analyzing the expression profile of 84 genes involved in cell cycle arrest, apoptosis, and DNA repair in HepG2 cells...
January 1, 2017: Toxicology and Industrial Health
https://www.readbyqxmd.com/read/28861953/fen1-promotes-tumor-progression-and-confers-cisplatin-resistance-in-non-small-cell-lung-cancer
#6
Lingfeng He, Libo Luo, Hong Zhu, Huan Yang, Yilan Zhang, Huan Wu, Hongfang Sun, Feng Jiang, Chandra S Kathera, Lingjie Liu, Ziheng Zhuang, Haoyan Chen, Feiyan Pan, Zhigang Hu, Jing Zhang, Zhigang Guo
No abstract text is available yet for this article.
September 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28854357/neil3-repairs-telomere-damage-during-s-phase-to-secure-chromosome-segregation-at-mitosis
#7
Jia Zhou, Jany Chan, Marie Lambelé, Timur Yusufzai, Jason Stumpff, Patricia L Opresko, Markus Thali, Susan S Wallace
Oxidative damage to telomere DNA compromises telomere integrity. We recently reported that the DNA glycosylase NEIL3 preferentially repairs oxidative lesions in telomere sequences in vitro. Here, we show that loss of NEIL3 causes anaphase DNA bridging because of telomere dysfunction. NEIL3 expression increases during S phase and reaches maximal levels in late S/G2. NEIL3 co-localizes with TRF2 and associates with telomeres during S phase, and this association increases upon oxidative stress. Mechanistic studies reveal that NEIL3 binds to single-stranded DNA via its intrinsically disordered C terminus in a telomere-sequence-independent manner...
August 29, 2017: Cell Reports
https://www.readbyqxmd.com/read/28749478/molecular-autopsy-in-maternal-fetal-medicine
#8
Hanan E Shamseldin, Wesam Kurdi, Fatima Almusafri, Maha Alnemer, Alya Alkaff, Zeneb Babay, Amal Alhashem, Maha Tulbah, Nada Alsahan, Rubina Khan, Bahauddin Sallout, Elham Al Mardawi, Mohamed Zain Seidahmed, Niema Meriki, Yasser Alsaber, Alya Qari, Ola Khalifa, Wafaa Eyaid, Zuhair Rahbeeni, Ahmed Kurdi, Mais Hashem, Tarfa Alshidi, Eman Al-Obeid, Firdous Abdulwahab, Niema Ibrahim, Nour Ewida, Karen El-Akouri, Mariam Al Mulla, Tawfeg Ben-Omran, Matthias Pergande, Sebahattin Cirak, Saeed Al Tala, Ranad Shaheen, Eissa Faqeih, Fowzan S Alkuraya
PurposeThe application of genomic sequencing to investigate unexplained death during early human development, a form of lethality likely enriched for severe Mendelian disorders, has been limited.MethodsIn this study, we employed exome sequencing as a molecular autopsy tool in a cohort of 44 families with at least one death or lethal fetal malformation at any stage of in utero development. Where no DNA was available from the fetus, we performed molecular autopsy by proxy, i.e., through parental testing.ResultsPathogenic or likely pathogenic variants were identified in 22 families (50%), and variants of unknown significance were identified in further 15 families (34%)...
July 27, 2017: Genetics in Medicine: Official Journal of the American College of Medical Genetics
https://www.readbyqxmd.com/read/28718810/dna2-an-important-player-in-dna-damage-response-or-just-another-dna-maintenance-protein
#9
REVIEW
Elzbieta Pawłowska, Joanna Szczepanska, Janusz Blasiak
The human DNA2 (DNA replication helicase/nuclease 2) protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER), interacting with the replication protein A (RPA) and the flap endonuclease 1 (FEN1). DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN) and Bloom syndrome protein (BLM)...
July 18, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28704715/differential-effect-of-the-overexpression-of-rad2-xpg-family-endonucleases-on-genome-integrity-in-yeast-and-human-cells
#10
Sonia Jimeno, Emilia Herrera-Moyano, Pedro Ortega, Andrés Aguilera
Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells...
July 3, 2017: DNA Repair
https://www.readbyqxmd.com/read/28685785/identification-of-human-flap-endonuclease-1-fen1-inhibitors-using-a-machine-learning-based-consensus-virtual-screening
#11
Amit Laxmikant Deshmukh, Sharat Chandra, Deependra Kumar Singh, Mohammad Imran Siddiqi, Dibyendu Banerjee
Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1...
July 25, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28678842/activation-of-dun1-in-response-to-nuclear-dna-instability-accounts-for-the-increase-in-mitochondrial-point-mutations-in-rad27-fen1-deficient-s-cerevisiae
#12
Aneta Kaniak-Golik, Renata Kuberska, Piotr Dzierzbicki, Ewa Sledziewska-Gojska
Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background...
2017: PloS One
https://www.readbyqxmd.com/read/28653660/phosphate-steering-by-flap-endonuclease-1-promotes-5-flap-specificity-and-incision-to-prevent-genome-instability
#13
Susan E Tsutakawa, Mark J Thompson, Andrew S Arvai, Alexander J Neil, Steven J Shaw, Sana I Algasaier, Jane C Kim, L David Finger, Emma Jardine, Victoria J B Gotham, Altaf H Sarker, Mai Z Her, Fahad Rashid, Samir M Hamdan, Sergei M Mirkin, Jane A Grasby, John A Tainer
DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions...
June 27, 2017: Nature Communications
https://www.readbyqxmd.com/read/28628639/small-molecule-inhibitors-uncover-synthetic-genetic-interactions-of-human-flap-endonuclease-1-fen1-with-dna-damage-response-genes
#14
Thomas A Ward, Peter J McHugh, Stephen T Durant
Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response...
2017: PloS One
https://www.readbyqxmd.com/read/28611278/lysine-methylation-of-fen1-by-set7-is-essential-for-its-cellular-response-to-replicative-stress
#15
Palaniraja Thandapani, Anthony M Couturier, Zhenbao Yu, Xing Li, Jean-François Couture, Shawn Li, Jean-Yves Masson, Stéphane Richard
The DNA damage response (DDR) is central to the cell survival and it requires post-translational modifications, in part, to sense the damage, amplify the signaling response and recruit and regulate DNA repair enzymes. Lysine methylation of histones such as H4K20 and non-histone proteins including p53 has been shown to be essential for the mounting of the DDR. It is well-known that the lysine methyltransferase SET7 regulates the DDR, as cells lacking this enzyme are hypersensitive to chemotherapeutic drugs. To define addition substrates of SET7 involved in the DDR, we screened a peptide array encompassing potential lysine methylation sites from >100 key DDR proteins and identified peptides from 58 proteins to be lysine methylated defining a methylation consensus sequence of [S>K-2; S>R-1; K0] consistent with previous findings...
May 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/28598207/the-relationship-between-single-nucleotide-polymorphisms-the-expression-of-dna-damage-response-genes-and-hepatocellular-carcinoma-in-a-polish-population
#16
Renata Krupa, Piotr Czarny, Paulina Wigner, Joanna Wozny, Maciej Jablkowski, Radzislaw Kordek, Janusz Szemraj, Tomasz Sliwinski
The molecular mechanism of hepatocellular carcinoma (HCC) is related to DNA damage caused by oxidative stress products induced by hepatitis B virus (HBV) or C (HCV) infection and exposure to environmental pollutants. Single-nucleotide polymorphisms (SNPs) of DNA damage response (DDR) genes may influence individual susceptibility to environmental risk factors and affect DNA repair efficacy, which, in turn, can influence the risk of HCC. The study evaluates a panel of 15 SNPs in 11 DDR genes (XRCC1, XRCC3, XPD, MUTYH, LIG1, LIG3, hOGG1, PARP1, NFIL1, FEN1, and APEX1) in 65 HCC patients, 50 HBV- and 50 HCV-infected non-cancerous patients, and 50 healthy controls...
August 2017: DNA and Cell Biology
https://www.readbyqxmd.com/read/28450457/roles-for-the-rad27-flap-endonuclease-in-mitochondrial-mutagenesis-and-double-strand-break-repair-in-saccharomyces-cerevisiae
#17
Prabha Nagarajan, Christopher T Prevost, Alexis Stein, Rachel Kasimer, Lidza Kalifa, Elaine A Sia
The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences...
June 2017: Genetics
https://www.readbyqxmd.com/read/28416706/the-gan-exonuclease-or-the-flap-endonuclease-fen1-and-rnase-hii-are-necessary-for-viability-of-thermococcus-kodakarensis
#18
Brett W Burkhart, Lubomira Cubonova, Margaret R Heider, Zvi Kelman, John N Reeve, Thomas J Santangelo
Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex...
July 1, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28373277/defining-the-rnaseh2-enzyme-initiated-ribonucleotide-excision-repair-pathway-in-archaea
#19
Margaret R Heider, Brett W Burkhart, Thomas J Santangelo, Andrew F Gardner
Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells...
May 26, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28371273/fen1-promotes-tumor-progression-and-confers-cisplatin-resistance-in-non-small-cell-lung-cancer
#20
Lingfeng He, Libo Luo, Hong Zhu, Huan Yang, Yilan Zhang, Huan Wu, Hongfang Sun, Feng Jiang, Chandra S Kathera, Lingjie Liu, Ziheng Zhuang, Haoyan Chen, Feiyan Pan, Zhigang Hu, Jing Zhang, Zhigang Guo
Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 (FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells...
June 2017: Molecular Oncology
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