DOCSLIB.ORG

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN Lawrence Berkeley National Laboratory Recent Work Title Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN. Permalink https://escholarship.org/uc/item/01s2z6gk Journal Molecular cell, 64(4) ISSN 1097-2765 Authors Stingele, Julian Bellelli, Roberto Alte, Ferdinand et al. Publication Date 2016-11-01 DOI 10.1016/j.molcel.2016.09.031 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Article Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN Graphical Abstract Authors Julian Stingele, Roberto Bellelli, Ferdinand Alte, ..., J. Mark Skehel, Michael Groll, Simon J. Boulton Correspondence [email protected] In Brief Stingele et al. discover the SPRTN metalloprotease to be crucial for DNA- protein crosslink repair in higher eukaryotes. In addition, several regulatory principles constraining SPRTN’s potentially toxic activity are described: a ubiquitin switch controlling chromatin access, a DNA switch triggering protease activity, and a negative feedback loop based on autocatalytic cleavage. Highlights Accession Numbers d The SPRTN metalloprotease repairs DNA-protein crosslinks 5JIG 5LN5 d A DNA switch controls SPRTN’s protease activity d A ubiquitin switch controls chromatin access of SPRTN d Structural insights reveal unique features of the SPRTN/Wss1 protease family Stingele et al., 2016, Molecular Cell 64, 688–703 November 17, 2016 ª 2016 The Author(s). Published by Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2016.09.031 Molecular Cell Article Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN Julian Stingele,1 Roberto Bellelli,1 Ferdinand Alte,2 Graeme Hewitt,1 Grzegorz Sarek,1 Sarah L. Maslen,3 Susan E. Tsutakawa,4 Annabel Borg,1 Svend Kjær,1 John A. Tainer,4,5 J. Mark Skehel,3 Michael Groll,2 and Simon J. Boulton1,6,* 1The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK 2Center for Integrated Protein Science at the Department Chemie, Lehrstuhl fur€ Biochemie, Technische Universitat€ Munchen,€ Lichtenbergstrasse 4, 85747 Garching, Germany 3MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK 4Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA 5Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA 6Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.09.031 SUMMARY and Bartek, 2009). DNA lesions range from abasic sites, small and bulky adducts, to single- and double-strand breaks, which Covalent DNA-protein crosslinks (DPCs) are toxic are repaired by lesion-specific and generally well-understood DNA lesions that interfere with essential chromatin mechanisms (Friedberg et al., 2014). However, specific repair transactions, such as replication and transcription. mechanisms for one particular type of lesion, covalent DNA-pro- Little was known about DPC-specific repair mecha- tein crosslinks (DPCs), have remained elusive. This is despite nisms until the recent identification of a DPC-pro- DPCs being extremely toxic as they directly block essential chro- cessing protease in yeast. The existence of a DPC matin transactions, such as replication and transcription (Fu et al., 2011; Nakano et al., 2012, 2013). protease in higher eukaryotes is inferred from data Xenopus laevis Covalent crosslinking of proteins to DNA can be caused by in egg extracts, but its identity re- various exogenous agents, such as ionizing radiation (IR), UV mains elusive. Here we identify the metalloprotease light, certain metal ions, and, importantly, platinum-based che- SPRTN as the DPC protease acting in metazoans. motherapeutics such as cisplatin and derivatives (Barker et al., Loss of SPRTN results in failure to repair DPCs and 2005; Stingele and Jentsch, 2015). Moreover, DPCs are induced hypersensitivity to DPC-inducing agents. SPRTN by endogenously produced reactive metabolites, such as form- accomplishes DPC processing through a unique aldehyde or acetaldehyde. Notably, formaldehyde is directly DNA-induced protease activity, which is controlled produced within chromatin as a by-product of the histone deme- by several sophisticated regulatory mechanisms. thylation reaction (Shi et al., 2004; Swenberg et al., 2011). Cellular, biochemical, and structural studies define Furthermore, abasic sites bear an aldehyde group that efficiently a DNA switch triggering its protease activity, a reacts with nucleosome proteins, thereby forming DPCs (Scze- panski et al., 2010). DPCs also are produced enzymatically by ubiquitin switch controlling SPRTN chromatin the entrapment of normally transient covalent DNA-protein reac- accessibility, and regulatory autocatalytic cleavage. tion intermediates of enzymes, such as topoisomerases 1 and 2 Our data also provide a molecular explanation (TOP1 and TOP2). Distortions within the DNA (e.g., caused by on how SPRTN deficiency causes the premature nearby DNA damage) or small molecules such as camptothecin aging and cancer predisposition disorder Ruijs-Aalfs (CPT) or etoposide inhibit religation and result in stable DPC syndrome. formation (Pommier, 2006). These enzymatic DPCs can be reversed by specific tyrosil-DNA phosphodiesterases (TDP1 and TDP2 acting on TOP1- and TOP2- adducts, respectively), INTRODUCTION which hydrolyze the covalent bond between the topoiso- merase’s active site residue and the DNA (Pommier et al., The integrity of DNA is constantly challenged by structural and 2014). Apart from these unique cases of enzymatic DPCs, it chemical alterations (Lindahl, 1993). Hence, restoration of the has been suggested that DPCs are generally repaired by canon- native DNA sequence and structure by damage-specific repair ical DNA repair pathways, such as nucleotide excision repair and mechanisms is essential to ensuring genome stability. Germline homologous recombination (Baker et al., 2007; de Graaf et al., mutations in crucial DNA repair enzymes result in several prema- 2009; Nakano et al., 2007, 2009). ture aging and cancer predisposition syndromes, highlighting Very recently a protease-based DPC repair mechanism was the fundamental importance of DNA repair in mammals (Jackson discovered in budding yeast (Stingele et al., 2014) and in 688 Molecular Cell 64, 688–703, November 17, 2016 ª 2016 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A Dvc-1 - C. elegans C PROTEASE PIP SHP SIM UBZ SPRTN - H. sapiens 100 224- 255- 270- 344- N2 PROTEASE SHP PIP 231 263 276 367 UBZ fcd-2 75 252- 325- 453- 261 332 475 50 % Viability% 25 dvc-1 PROTEASE fcd-2; dvc-1 SHP VIM SIM SIM 0 Wss1 - S. cerevisiae 02468 152- 208- 247- 265- Formaldehyde [mM] 160 218 252 269 B D 100 100 N2 100 N2 75 75 75 N2 50 50 50 fcd-2 % Viability % Viability % dvc-1 25 25 Viability % 25 dvc-1 dvc-1 0 0 0 fcd-2; dvc-1 02468 0 200 400 ctrl 24 48 72 Formaldehyde [mM] Cisplatin [μM] 200 μM Cisplatin [h] E 100 100 100 N2 xpa-1(RNAi) 75 75 75 N2 50 50 dvc-1 50 N2 % Viability % Viability % 25 dvc-1 25 Viability% 25 dvc-1 xpa-1(RNAi); dvc-1 0 0 0 0 70 140 070140 02468 IR [Gy] UVC [J/m2] Formaldehyde [mM] FG**** H 300 100 100 N2 100 him-6 N2 75 75 200 50 50 50 dvc-1 100 Brood sizeBrood % Viability % 25 Viability % 25 him-6 % Viable% progeny dvc-1 him-6; dvc-1 0 0 0 0 02468 0 100 200 300 400 N2 N2 Formaldehyde [mM] Cisplatin [μM] dvc-1him-6 dvc-1him-6 him-6; dvc-1 him-6; dvc-1 Figure 1. SPRTN/Dvc-1 Provides Resistance toward DPC-Inducing Agents in Worms and Operates Independently of FANCD2/Fcd-2 and Parallel to BLM/Him-6 (A) Domain structures and evolutionary distances of the protease domain of SPRTN/Wss1 protease family members of humans, worms, and budding yeast. SPRTN/Wss1 proteases bear interaction domains for p97/Cdc48 (SHP-box, VIM), recognition modules for ubiquitin (UBZ) or SUMO (SIM), and in metazoansa PCNA-interaction motif (PIP-box). (B) C. elegans mutant strains lacking functional SPRTN (dvc-1) are specifically sensitive to the DPC-inducing agents. Formaldehyde sensitivity was determined in synchronized L1 larvae. Cisplatin, UVC light, and IR sensitivities were assessed by measuring embryonic survival of progeny after exposure of adult animals. Error bars indicate SEM of –two to four independent experiments. (C) FANCD2 is not involved in providing formaldehyde resistance in synchronized L1 larvae. Error bars indicate SEM of two independent experiments. (D) FANCD2 provides resistance to chronic cisplatin exposure by a mechanism distinct to DPC repair by SPRTN. Viability was assessed by determination of embryonic survival of progeny of young adult animals kept on cisplatin-containing plates (200 mM) for the indicated amount of time. Error bars indicate SEM of two independent experiments. (E) Loss of XPA does not result in increased formaldehyde sensitivity in synchronized L1 worms. Error bars indicate SEM of two independent experiments. (legend continued on next page) Molecular Cell 64, 688–703, November 17, 2016 689 Xenopus laevis egg extracts (Duxin et al., 2014). In yeast, DPC assessing sensitivity to DPC-inducing agents. DNA damage proteolysis is catalyzed by the metalloprotease Wss1, which sensitivity is typically measured in worms by treating young adult permits replication in the presence of DPCs and provides resis- animals, followed by determining viability of their progeny as a tance toward DPC-inducing agents. Intriguingly, Wss1 is a DNA- proxy for repair defects in the germline, which is the only prolif- dependent protease that degrades DNA-bound substrates erating tissue in adult animals (Figure S1A). However, this treat- in vitro irrespective of identity. Importantly, in Xenopus egg ex- ment regimen is not suitable for testing formaldehyde sensitivity, tracts, a DPC-containing plasmid is repaired by a similar mech- because treated adult worms succumb to death at doses that anism, indicating that protease-based DPC repair is conserved.
Load more

Recommended publications
  • DNA Topoisomerases and Cancer Topoisomerases and TOP Genes in Humans Humans Vs
    DNA Topoisomerases Review DNA Topoisomerases And Cancer Topoisomerases and TOP Genes in Humans Humans vs. Escherichia Coli Topoisomerase differences Comparisons Topoisomerase and genomes Top 1 Top1 and Top2 differences Relaxation of DNA Top1 DNA supercoiling DNA supercoiling In the context of chromatin, where the rotation of DNA is constrained, DNA supercoiling (over- and under-twisting and writhe) is readily generated. TOP1 and TOP1mt remove supercoiling by DNA untwisting, acting as “swivelases”, whereas TOP2a and TOP2b remove writhe, acting as “writhases” at DNA crossovers (see TOP2 section). Here are some basic facts concerning DNA supercoiling that are relevant to topoisomerase activity: • Positive supercoiling (Sc+) tightens the DNA helix whereas negative supercoiling (Sc-) facilitates the opening of the duplex and the generation of single-stranded segments. • Nucleosome formation and disassembly absorbs and releases Sc-, respectively. • Polymerases generate Sc+ ahead and Sc- behind their tracks. • Excess of Sc+ arrests DNA tracking enzymes (helicases and polymerases), suppresses transcription elongation and initiation, and destabilizes nucleosomes. • Sc- facilitates DNA melting during the initiation of replication and transcription, D-loop formation and homologous recombination and nucleosome formation. • Excess of Sc- favors the formation of alternative DNA structures (R-loops, guanine quadruplexes, right-handed DNA (Z-DNA), plectonemic structures), which then absorb Sc- upon their formation and attract regulatory proteins. The
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Deoxyguanosine Depurination and Yield DNA–Protein Cross-Links in Nucleosome Core Particles and Cells
    Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells Kun Yanga, Daeyoon Parkb, Natalia Y. Tretyakovac, and Marc M. Greenberga,1 aDepartment of Chemistry, Johns Hopkins University, Baltimore, MD 21218; bDepartment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Twin Cities, Minneapolis, MN 55417; and cDepartment of Medicinal Chemistry, University of Minnesota, Twin Cities, Minneapolis, MN 55417 Edited by Cynthia J. Burrows, University of Utah, Salt Lake City, UT, and approved October 18, 2018 (received for review August 6, 2018) Monofunctional alkylating agents preferentially react at the adducts. DPC formation between histone proteins and MdG was N7 position of 2′-deoxyguanosine in duplex DNA. Methylated also observed in MMS-treated V79 Chinese hamster lung cells. DNA, such as that produced by methyl methanesulfonate (MMS) Intracellular DPC formation from MdG could have significant and temozolomide, exists for days in organisms. The predominant biological effects. consequence of N7-methyl-2′-deoxyguanosine (MdG) is widely be- Compared with other DNA lesions, MdG minimally perturbs lieved to be abasic site (AP) formation via hydrolysis, a process duplex structure (12). The steric effects of N7-methylation within that is slow in free DNA. Examination of MdG reactivity within the major groove are minimal, and the aromatic planar structure nucleosome core particles (NCPs) provided two general observa- is maintained. Although the Watson–Crick hydrogen bonding tions. MdG depurination rate constants are reduced in NCPs com- pattern of dG is retained, recent structural studies raise the pared with when the identical DNA sequence is free in solution.
    [Show full text]
  • Table SI. Genes Upregulated ≥ 2-Fold by MIH 2.4Bl Treatment Affymetrix ID
    Table SI. Genes upregulated 2-fold by MIH 2.4Bl treatment Fold UniGene ID Description Affymetrix ID Entrez Gene Change 1558048_x_at 28.84 Hs.551290 231597_x_at 17.02 Hs.720692 238825_at 10.19 93953 Hs.135167 acidic repeat containing (ACRC) 203821_at 9.82 1839 Hs.799 heparin binding EGF like growth factor (HBEGF) 1559509_at 9.41 Hs.656636 202957_at 9.06 3059 Hs.14601 hematopoietic cell-specific Lyn substrate 1 (HCLS1) 202388_at 8.11 5997 Hs.78944 regulator of G-protein signaling 2 (RGS2) 213649_at 7.9 6432 Hs.309090 serine and arginine rich splicing factor 7 (SRSF7) 228262_at 7.83 256714 Hs.127951 MAP7 domain containing 2 (MAP7D2) 38037_at 7.75 1839 Hs.799 heparin binding EGF like growth factor (HBEGF) 224549_x_at 7.6 202672_s_at 7.53 467 Hs.460 activating transcription factor 3 (ATF3) 243581_at 6.94 Hs.659284 239203_at 6.9 286006 Hs.396189 leucine rich single-pass membrane protein 1 (LSMEM1) 210800_at 6.7 1678 translocase of inner mitochondrial membrane 8 homolog A (yeast) (TIMM8A) 238956_at 6.48 1943 Hs.741510 ephrin A2 (EFNA2) 242918_at 6.22 4678 Hs.319334 nuclear autoantigenic sperm protein (NASP) 224254_x_at 6.06 243509_at 6 236832_at 5.89 221442 Hs.374076 adenylate cyclase 10, soluble pseudogene 1 (ADCY10P1) 234562_x_at 5.89 Hs.675414 214093_s_at 5.88 8880 Hs.567380; far upstream element binding protein 1 (FUBP1) Hs.707742 223774_at 5.59 677825 Hs.632377 small nucleolar RNA, H/ACA box 44 (SNORA44) 234723_x_at 5.48 Hs.677287 226419_s_at 5.41 6426 Hs.710026; serine and arginine rich splicing factor 1 (SRSF1) Hs.744140 228967_at 5.37
    [Show full text]
  • Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass
    cancers Review Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass Nicole A. Wilkinson y, Katherine S. Mnuskin y, Nicholas W. Ashton * and Roger Woodgate * Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, USA; [email protected] (N.A.W.); [email protected] (K.S.M.) * Correspondence: [email protected] (N.W.A.); [email protected] (R.W.); Tel.: +1-301-435-1115 (N.W.A.); +1-301-435-0740 (R.W.) Co-first authors. y Received: 29 August 2020; Accepted: 29 September 2020; Published: 2 October 2020 Simple Summary: Ubiquitin and ubiquitin-like proteins are conjugated to many other proteins within the cell, to regulate their stability, localization, and activity. These modifications are essential for normal cellular function and the disruption of these processes contributes to numerous cancer types. In this review, we discuss how ubiquitin and ubiquitin-like proteins regulate the specialized replication pathways of DNA damage bypass, as well as how the disruption of these processes can contribute to cancer development. We also discuss how cancer cell survival relies on DNA damage bypass, and how targeting the regulation of these pathways by ubiquitin and ubiquitin-like proteins might be an effective strategy in anti-cancer therapies. Abstract: Many endogenous and exogenous factors can induce genomic instability in human cells, in the form of DNA damage and mutations, that predispose them to cancer development. Normal cells rely on DNA damage bypass pathways such as translesion synthesis (TLS) and template switching (TS) to replicate past lesions that might otherwise result in prolonged replication stress and lethal double-strand breaks (DSBs).
    [Show full text]
  • Characterization of SPRTN, the First Mammalian Metalloprotease That Repairs DNA-Protein-Crosslinks
    Aus dem Fachbereich Medizin der Johann Wolfgang Goethe-Universität Frankfurt am Main betreut am Gustav Embden-Zentrum der Biochemie Institut für Biochemie II (Kardiovaskuläre Biochemie) Direktor: Prof. Dr. Ivan Dikic Characterization of SPRTN, the first mammalian metalloprotease that repairs DNA-protein-crosslinks Dissertation zur Erlangung des Doktorgrades der Medizin des Fachbereichs Medizin der Johann Wolfgang Goethe-Universität Frankfurt am Main vorgelegt von Stefan Prgomet, dr. med. aus Düsseldorf Frankfurt am Main, 2019 1 Dekan: Prof. Dr. Stefan Zeuzem Referent/in: Prof. Dr. Ivan Dikic Korreferent/in: Prof. Dr. Jörg Trojan Tag der mündlichen Prüfung: 12.05.2020 2 Contents 1 Summary / Zusammenfassung .................................................................... 6 / 8 2 Introduction ...................................................................................................... 10 2.1 SPRTN regulates a progeria and tumorigenesis axis ............................ 10 2.1.1 RJALS-syndrome as the clinical manifestation of SPRTN malfunction 10 2.1.2 SPRTN acts in DNA damage repair ..................................................... 11 2.1.3 SPRTN’s essential function remains unknown ..................................... 13 2.2 DNA damage response (DDR) .................................................................. 16 2.2.1 Types of DNA-lesions ........................................................................... 16 2.2.2 DNA damage repair mechanisms ......................................................... 18 2.2.3
    [Show full text]
  • 1 USP11 Deubiquitinates Monoubiquitinated SPRTN to Repair DNA-Protein Crosslinks Megan Perry,1 Sai Sundeep Kollala,1 Meghan Bieg
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.30.180471; this version posted July 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. USP11 deubiquitinates monoubiquitinated SPRTN to repair DNA-protein crosslinks Megan Perry,1 Sai Sundeep Kollala,1 Meghan Biegert,1 Grace Su,2 Manohar Kodavati,3 Halle Mallard,1 Natasha Kreiling,1 Alexander Holbrook,1 and Gargi Ghosal1,4* 1Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198 2Department of Biology, Doane University, Crete, NE 68333 3Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030 4Fred and Pamela Buffett Cancer Center, Omaha NE 68105 *Correspondence: [email protected] SUMMARY DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription and recombination. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN chromatin accessibility during DPC repair. The deubiquitinase regulating SPRTN function in DPC repair is unknown. Here, we identify USP11 as a SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC- inducing agents. Our findings elucidate the function of USP11 in the regulation of SPRTN monoubiquitination and SPRTN-mediated DPC repair.
    [Show full text]
  • Spartan Deficiency Causes Genomic Instability and Progeroid Phenotypes
    ARTICLE Received 25 Jun 2014 | Accepted 4 Nov 2014 | Published 11 Dec 2014 DOI: 10.1038/ncomms6744 OPEN Spartan deficiency causes genomic instability and progeroid phenotypes Reeja S. Maskey1,*, Myoung Shin Kim2,*, Darren J. Baker3, Bennett Childs1, Liviu A. Malureanu1, Karthik B. Jeganathan1, Yuka Machida2, Jan M. van Deursen1 & Yuichi J. Machida2,4 Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria. 1 Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. 2 Department of Oncology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. 3 Department of Pediatric and Adolescent Medicine, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. 4 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.
    [Show full text]
  • Arp58546 P050
    Aviva Systems Biology TUFM antibody - middle region (ARP58546_P050) Product Number ARP58546_P050 Product Page http://www.avivasysbio.com/tufm-antibody-middle-region-arp58546-p050.html Product Name TUFM antibody - middle region (ARP58546_P050) Size 100 ul Gene Symbol TUFM Alias Symbols COXPD4, EF-TuMT, EFTU, P43 Protein Size (# AA) 455 amino acids Molecular Weight 50kDa Product Format Liquid. Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. NCBI Gene Id 7284 Host Rabbit Clonality Polyclonal Concentration Batch dependent within range: 100 ul at 0.5 - 1 mg/ml Official Gene Full Tu translation elongation factor, mitochondrial Name Description This is a rabbit polyclonal antibody against TUFM. It was validated on Western Blot using a cell lysate as a positive control. Aviva Systems Biology strives to provide antibodies covering each member of a whole protein family of your interest. We also use our best efforts to provide you antibodies recognize various epitopes of a target protein. For availability of antibody needed for your experiment, please inquire ([email protected]). Peptide Sequence Synthetic peptide located within the following region: PEKELAMPGEDLKFNLILRQPMILEKGQRFTLRDGNRTIGTGLVTNTLAM Target Reference Valente,L., (2007) Am. J. Hum. Genet. 80 (1), 44-58 Description of TUFM is a protein which participates in protein translation in mitochondria. Mutations in Target this gene have been associated with combined oxidative phosphorylation deficiency resulting in lactic acidosis and fatal encephalopathy. This gene encodes a protein which participates in protein translation in mitochondria. Mutations in this gene have been associated with combined oxidative phosphorylation deficiency resulting in lactic acidosis and fatal encephalopathy.
    [Show full text]
  • The Role of P97 Segregase in the Repair of Dnaprotein Crosslink In
    The role of P97 segregase in the repair of DNA­protein crosslink in vivo using CRISPR/Cas9 gene editing in zebrafish P­02.3­26 C. OttenI, C. SupinaI, M. PopovicI IRuder Boskovic Institute, Zagreb, Croatia DNA­Protein­Crosslinks (DPCs) are DNA lesions which occur when proteins become irreversibly covalently bound to DNA, thereby physically blocking all DNA transactions including chromatin remodeling, replication, transcription and repair. DPC repair (DPCR) is a specialized DNA damage repair pathway in which crosslinked proteins are proteolytically or nucleolytically cleaved. Recently, the specific roles of several enzymes in DPCR have been uncovered, and their dysfunction has been linked to ageing, cancer and neurodegenerative diseases. To quantify DPCs, we are developing a new method using stably transfected HeLa cells expressing a GFP­tagged Histone which can form model DPCs upon e.g. formaldehyde treatment. The presence of GFP in particular cellular fractions, which depends on the presence and activity of DPCR enzymes, can then be analyzed, either by fluorescence or by western blot. Once the method is established in cell culture, we aim to develop an in vivo assay in zebrafish. Currently, we are creating zebrafish mutants for DPCR factors using Cripsr/Cas9 gene editing with the aim of studying the function of target proteins in DPCR in vivo during embryonic development and in adults. In particular, we are generating a zebrafish mutant line by introducing the K524A mutation in the p97 gene using the CRISPR/Cas9 knock­in method to elucidate the role of the segregase P97 specifically in DPCR. We will investigate the epistasis of P97 with Sprtn, which is a central component of DPCR and for which we are generating equivalent mutant zebrafish lines.
    [Show full text]
  • The Genetics of Physiological Dispersion in Signs of Diabetes Using Murine Models
    The Genetics of Physiological Dispersion in Signs of Diabetes Using Murine Models Dayna Brown Department of Biology University of Prince Edward Island Charlottetown, PEI, Canada A thesis submitted in partial fulfilment of the requirements of the Honours Programme in the Department of Biology This Thesis is ___________________________________ Accepted Dean of Science University of Prince Edward Island May 2018 ii ABSTRACT Diabetes is a collection of diseases that affects hundreds of millions of people, with type 2 (T2D) being the most prevalent. The heritability of T2D has yet to be explained, despite many studies. This study aims to determine a piece of the missing heritability by using the newly emerging phenomenon of ‘phenotypic dispersion’ (PD) which refers to genes affecting not only mean effects but also the residual variance within genotypes. The association of loci to residual variance of traits is the method used to determine genes that may contribute to T2D development. Eight in silico mouse data sets were analyzed to test for relationships between mapped microsatellite markers and the PD of diabetic plasma variables (cholesterol, high and low-density lipoprotein, and triglyceride). 29 loci were found to be significantly associated with PD of traits within the data sets. Single nucleotide polymorphisms (SNPs) in genes nearby the associated loci were identified, with Fto, Apoa2, Foxa2, Bpifb6, and Apt1a4 being the most notable genes involved. The association between genotype and PD at all associated loci showed that when the genotype was dominant that it was dominant for low dispersion, which is suggestive of a genetic mechanism controlling the dispersion of traits.
    [Show full text]
  • TEX264 Coordinates P97- and SPRTN-Mediated Resolution of Topoisomerase 1-DNA Adducts
    This is a repository copy of TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/158341/ Version: Published Version Article: Fielden, J., Wiseman, K., Torrecilla, I. et al. (14 more authors) (2020) TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nature Communications, 11 (1). 1274. ISSN 2041-1723 https://doi.org/10.1038/s41467-020-15000-w Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ ARTICLE https://doi.org/10.1038/s41467-020-15000-w OPEN TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts John Fielden1,7, Katherine Wiseman1,7, Ignacio Torrecilla 1, Shudong Li1, Samuel Hume 1, Shih-Chieh Chiang2, Annamaria Ruggiano1, Abhay Narayan Singh1, Raimundo Freire 3,4,5, Sylvana Hassanieh1, Enric Domingo 1, Iolanda Vendrell1,6, Roman Fischer 6, Benedikt M. Kessler 6, Timothy S. Maughan1, ✉ Sherif F. El-Khamisy 2 & Kristijan Ramadan 1 1234567890():,; Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA repli- cation and transcription.
    [Show full text]