DOCSLIB.ORG

Relaxase (Tral) of Incpa Plasmid RP4 Catalyzes a Site-Specific

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Relaxase (Tral) of Incpa Plasmid RP4 Catalyzes a Site-Specific Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2925-2929, April 1993 Biochemistry Relaxase (Tral) of IncPa plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA (bacterial conjugation/active-site tyrosine/sequence-specific recognition of single-stranded DNA/termination of transfer DNA replication) WERNER PANSEGRAUt, WERNER SCHRODERt, AND ERICH LANKAt§ tMax-Planck-Institut fur Molekulare Genetik, Abteilung Schuster, Ihnestrasse 73, D-1000 Berlin 33, Federal Republic of Germany; and tFreie Universitat Berlin, Institut fur Biochemie, Fabeckstrasse 36a, D-1000 Berlin 33, Federal Republic of Germany Communicated by F. William Studier, December 14, 1992 ABSTRACT Conjugative DNA transfer of the self- (TraI) to nic. The role of TraH is to stabilize the initial transmissible broad-host-range plasmid RP4 is initiated by complex of form I oriT DNA, TraJ, and TraI by specific strand- and site-specific cleavage at the nick site (nic) of the protein-protein interactions (2). transfer origin (oriT). Cleavage results in covalent attachment We report on the mechanism of site- and strand-specific of the plasmid-encoded relaxase (TraI) to the 5'-terminal cleavage at oriT as part of a study of components involved in 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the initiation and termination processes of RP4-mediated the center of the catalytic site of TraI, mediating cleavage via DNA transfer. The specific interaction of RP4 relaxase with formation of a phosphodiester between the DNA 5' phosphoryl defined oligodeoxyribonucleotide substrates served to de- and the aromatic hydroxyl group. The specificity of cleavage scribe (i) the cleavage and rejoining reaction, (ii) the nucle- seen with form I oriT DNA was verified with short oligodeoxy- otide specificity within the nick region, and (iii) the nature of ribonucleotides embracing the nick region. The reaction re- the covalent bond between the DNA and the relaxase. To test quires TraI and Mg2+ but is independent of the relaxosome the hypothesis that termination of transfer DNA replication component TraJ. Cleavage produces one oligonucleotide frag- occurs by site-specific recombination between single- ment with a free 3' hydroxyl, the other part forms a covalent stranded oriT sites, we employed purified TraI of plasmid Tral-oligonucleotide adduct. Like nicking of form I oniT DNA, RP4 in an in vitro assay detecting strand exchange at nic. Tral-catalyzed oligonucleotide cleavage reaches an equilibrium Consideration of analogous DNA sequences in a variety of when about 30% of the input Tral exists as a covalent protein- other DNA transfer systems, including the agrobacterial Ti DNA complex. In the presence of two differently sized oligo- plasmids, leads us to suggest that the mechanism described nucleotides, defined hybrid oligonucleotides are produced, here is a widespread mode ofgene transmission between both demonstrating that TraI catalyzes recombination of two single closely and remotely related organisms. strands at nic. This rmding shows that TraI possesses cleaving- joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 MATERIALS AND METHODS nucleotides adjacent to nic. Only certain base changes in a few Proteins and DNA. TraI and TraIA3 were purified as positions are tolerated, whereas the sequence of the 5' terminal were labeled either nucleotides apparently is irrelevant for recognition by Tral. described (2). Oligodeoxyribonucleotides The reactions described here further support the hypothesis at their 3' ends by using [a-32P]ddATP (3000 Ci/mmol; 1 Ci that DNA transfer via conjugation involves a roiling circle-like = 37 GBq) and terminal deoxynucleotidyltransferase (Am- mechanism which generates the immigrant single strand while ersham) or at their 5'-ends by using [y-32P]ATP (3000 Ci/ DNA-bound TraI protein scans for the occurrence of a second mmol) and phage T4 polynucleotide kinase (4). cleavage site at the donor-recipient interface. Cleavage and Site-Specific Recombination of Oligodeoxyri- bonucleotides by TraL Oligodeoxyribonucleotides (5 pmol) were incubated with Tral (4 ,ug, 50 pmol) in 20 ,ul of TNM (20 Horizontal gene transfer by bacterial conjugation requires the mM Tris HCl, pH 8.8/50 mM NaCl/5 mM MgCl2) for 3 hr at generation of a defined DNA single strand by a rolling 370C. When appropriate, the reaction was stopped by the circle-type mechanism of replication. Following relaxosome K and formation, the key step in this process is site-specific hydro- addition ofSDS (10 mg/ml) and proteinase (100 ,Ag/ml) lysis of a scissile phosphodiester bond at oriTby the relaxase, incubation for an additional 30 min at 37°C. Products were which becomes covalently attached to the 5' phosphoryl end analyzed in a 20% (wt/vol) polyacrylamide gel containing 8 ofthe DNA (for review see ref. 1). Covalent adduct formation M urea. Reaction products were quantified by autoradiogra- is thought to conserve the bond energy required for recircu- phy of gels with the storage phosphor technology (5). larization of the transferred strand by a site-specific recom- Sequencing the Active Site of TraL Preparation of radio- bination event at the nick site of oriT. Use of the IncPa labeled substrate. Oligodeoxyribonucleotide (17-mer, 5'- plasmid RP4 as a model system for studying the molecular TTCACCTATCCTGCCCG-3', 1 nmol) was labeled at the 3' mechanisms of DNA processing during conjugation showed end with [a-32P]dATP (3.3 pmol; 3000 Ci/mmol). [a-32P]- that all genes required for relaxosome formation are clustered dATP was used instead of [a-32P]ddATP to avoid different adjacent to the intergenic oriT (2). During assembly of RP4 separation properties of labeled and unlabeled oligonucle- relaxosomes, the proteins TraJ, TraI, and TraH interact at otide-peptide adducts during HPLC. Due to the great molar oriT in a cascade-like mechanism preparing the superhelical excess of oligonucleotide over the labeled nucleotide, the plasmid for initiation of transfer DNA replication (2). The majority of radiolabeled oligonucleotides were 18-mers car- application ofpurified components in vitro revealed that TraJ rying only one 3' [32P]dAMP. The labeled 18-mer was isolated is a specific oriT-binding protein (3) that directs the relaxase by preparative TLC using the United States Biochemical SurePure system. To obtain an amount of oligonucleotide suitable for preparative cleavage by TraI, unlabeled 18-mer The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 2925 Downloaded by guest on October 7, 2021 2926 Biochemistry: Pansegrau et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5'-TTCACCTATCCTGCCCGA-3' was added in 200-fold ex- A cess. Formation of covalent TraI-oligonucleotide adduct. One hundred sixty nanomoles of the mixture of labeled and -18-mer unlabeled 18-mer was incubated with 80 nmol of TraIA3 in 2 = = _-~~~~17-mer by ml TNM for 3 hr at 37°C. The reaction was stopped - d(pCCCG)ddA- addition of SDS (10 mg/ml) and proteinase K (100 ,ug/ml), . ....... ..... .- 13-mer and incubation was continued for 1 hr. After lyophilization, _PePPOK- the reaction mixture was dissolved in 0.5 ml of water. After centrifugation for 15 min at 20,000 x g and 0WC, the super- natant was applied to a C18 reversed-phase column (4 x 250 a b c d e f (pH mm) equilibrated with 10 mM triethylammonium acetate 5' 5' 5' 3' 3' 3' label 6.5). Peptides were eluted over a period of 3 hr at 0.8 ml/min - + + - + + Tral with a linear gradient of 6-30% acetonitrile in 10 mM trieth- - - + - - + proteinase K ylammonium acetate (pH 6.5). Eluted peptides were detected by A220. Fractions containing pepproK-d(pCCCGA*) were B identified (i) by measuring radioactivity and (ii) by gel n4) electrophoresis. pepPrOK-d(pCCCGA*) was eluted at 18.5% acetonitrile (pepproK means a proteinase K-generated Tral ov peptide with a few amino acid residues; an asterisk indicates n 20 the presence of a [32P]phosphoryl group). One and a half 00z'o 10 vOia nanomoles was obtained. The peak fraction (750 pmol) was 0. analyzed on an Applied Biosystems model 473A sequencer 4) (6). 10 20 RESULTS molar ratio 118-mer]/LTral] of by TraL. Muta- Site-Specific Cleavage Oligonucleotides FIG. 2. Site-specific cleavage of single-stranded oligonucleotides tions in the nick region of plasmid RP4 (Fig. 1) demonstrated by Tral. (A) Tral was incubated with oligonucleotides and reactions that several positions in the 8-bp stretch between the TraJ were stopped and analyzed as described under Materials and Meth- binding region and nic are crucial to Tral-mediated cleavage ods. Presence or absence of Tral in the reactions is shown. Where (7). This indicated that other nucleotides in addition to nic indicated, the cleavage reaction was followed by proteinase K diges- interact with the relaxase. It is expected, therefore, that tion. Oligonucleotides were 32P-labeled at their 3' or 5' end. Positions recognition of oriT sequences by Tral is not exclusively of bands corresponding to the input oligonucleotides [17-mer with mediated by the TraJ-oriT complex. It is also conceivable that 5'-end label, d(p*TTCACCTATCCTGVCCCG), and 18-mer with binding of TraK in this region changes the local DNA 3'-end label, d(TTCACCTATCCTGVCCCG)ddA*] and cleavage that nic is exposed as a single strand for cleavage products [13-mer with 5'-end label, d(p*TTCACCTATCCTG), or topology so pepproK-d(pCCCG)ddA* with 3'-end label] are indicated at right. (B) by Tral (8). Tral was incubated with various amounts of 3'-end-labeled 18-mer as To test the hypothesis of DNA single-strand cleavage, described above.
Load more

Recommended publications
  • Up-Regulation of Telomerase Activity in Human Pancreatic Cancer Cells After Exposure to Etoposide
    British Journal of Cancer (2000) 82(11), 1819–1826 © 2000 Cancer Research Campaign DOI: 10.1054/ bjoc.2000.1117, available online at http://www.idealibrary.com on Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide N Sato, K Mizumoto, M Kusumoto, S Nishio, N Maehara, T Urashima, T Ogawa and M Tanaka Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Summary Telomerase plays a critical role in the development of cellular immortality and oncogenesis. Activation of telomerase occurs in a majority of human malignant tumours, and the relation between telomerase and vulnerability to drug-mediated apoptosis remains unclear. In this study, we demonstrate, for the first time, up-regulation of telomerase activity in human pancreatic cancer cells treated with etoposide, a topoisomerase II inhibitor. Exposure of MIA PaCa-2 cells to etoposide at various concentrations (1–30 µM) resulted in two- to threefold increases in telomerase activity. Up-regulation was detectable 24 h after drug exposure and was accompanied by enhanced expression of mRNA of the human telomerase reverse transcriptase. Telomerase activation was also observed in AsPC-1 and PANC-1 cells but not in KP-3 and KP-1N cells. Furthermore, we found a negative correlation between increased telomerase activity and the percentage of dead cells after etoposide treatment. These findings suggest the existence of an anti-apoptotic pathway through which telomerase is up-regulated in response to DNA damage. This telomerase activation pathway may be one of the mechanisms responsible for the development of etoposide resistance in certain pancreatic cancer cells.
    [Show full text]
  • (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution Debayan Purkait†A, Debolina Bandyopadhyay†A, and Padmaja P
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293639; this version posted September 11, 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. Vital Insights into Prokaryotic Genome Compaction by Nucleoid-Associated Protein (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution Debayan Purkait†a, Debolina Bandyopadhyay†a, and Padmaja P. Mishra*a aChemical Sciences Division, Saha Institute of Nuclear Physics, India aHomi Bhaba National Institute (HBNI), India †Both the authors have contributed equally. * Corresponding author Abstract Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a ‘Slacked-Dynamic’ state throughout the observation window where many of them have switched between multiple ‘Wobbling States’ in the course of attainment of packaged form. A range of DNA ‘Flexure Angles’ has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes.
    [Show full text]
  • Cell Cycle Regulation by the Bacterial Nucleoid
    Available online at www.sciencedirect.com ScienceDirect Cell cycle regulation by the bacterial nucleoid David William Adams, Ling Juan Wu and Jeff Errington Division site selection presents a fundamental challenge to all terminate in the terminus region (Ter; 1808). Once organisms. Bacterial cells are small and the chromosome chromosome replication and segregation are complete (nucleoid) often fills most of the cell volume. Thus, in order to the cell is ready to divide. In Bacteria this normally occurs maximise fitness and avoid damaging the genetic material, cell by binary fission and in almost all species this is initiated division must be tightly co-ordinated with chromosome by the assembly of the tubulin homologue FtsZ into a replication and segregation. To achieve this, bacteria employ a ring-like structure (‘Z-ring’) at the nascent division site number of different mechanisms to regulate division site (Figure 1) [1]. The Z-ring then functions as a dynamic selection. One such mechanism, termed nucleoid occlusion, platform for assembly of the division machinery [2,3]. Its allows the nucleoid to protect itself by acting as a template for central role in division also allows FtsZ to serve as a nucleoid occlusion factors, which prevent Z-ring assembly over regulatory hub for the majority of regulatory proteins the DNA. These factors are sequence-specific DNA-binding identified to date [2,4]. Nevertheless, the precise ultra- proteins that exploit the precise organisation of the nucleoid, structure of the Z-ring and whether or not it plays a direct allowing them to act as both spatial and temporal regulators of role in force-generation during division remains contro- bacterial cell division.
    [Show full text]
  • Catalytic Domain of Plasmid Pad1 Relaxase Trax Defines a Group Of
    Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases María Victoria Franciaa,1, Don B. Clewellb,c, Fernando de la Cruzd, and Gabriel Moncaliánd aServicio de Microbiología, Hospital Universitario Marqués de Valdecilla e Instituto de Formación e Investigación Marqués de Valdecilla, Santander 39008, Spain; bDepartment of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109; cDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and dDepartamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria–Consejo Superior de Investigaciones Científicas–Sociedad para el Desarrollo Regional de Cantabria, Santander 39011, Spain Edited by Roy Curtiss III, Arizona State University, Tempe, AZ, and approved July 9, 2013 (received for review May 30, 2013) Plasmid pAD1 is a 60-kb conjugative element commonly found in known relaxases show two characteristic sequence motifs, motif I clinical isolates of Enterococcus faecalis. The relaxase TraX and the containing the catalytic Tyr residue (which covalently attaches to primary origin of transfer oriT2 are located close to each other and the 5′ end of the cleaved DNA) and motif III with the His-triad have been shown to be essential for conjugation. The oriT2 site essential for relaxase activity (facilitating the cleavage reaction contains a large inverted repeat (where the nic site is located) by activation of the catalytic Tyr). This His-triad has been used as adjacent to a series of short direct repeats. TraX does not show a relaxase diagnostic signature (12). fi any of the typical relaxase sequence motifs but is the prototype of Plasmid pAD1 relaxase, TraX, was identi ed (9) and shown to oriT2 a unique family of relaxases (MOB ).
    [Show full text]
  • Relaxation Complexes of Plasmids Colel and Cole2:Unique Site of The
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 3854-3857, October 1974 Relaxation Complexes of Plasmids ColEl and ColE2: Unique Site of the Nick in the Open Circular DNA of the Relaxed Complexes (Escherichia coli/supercoiled DNA/endonuclease/restriction enzyme/DNA replication) MICHAEL A. LOVETT, DONALD G. GUINEY, AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, Calif. 92037 Communicated by Stanley L. Miller, June 26, 1974 ABSTRACT The product of the induced relaxation of volved in the production of a single-strand cleavage in the supercoiled DNA-protein relaxation complexes of colicino- initiation of replication and/or conjugal transfer of plasmid genic factors El (ColEl) and E2 (CoIE2) is an open circular DNA molecule with a strand-specific nick. Cleavage of the DNA. An involvement in such processes would most likely open circular DNA of each relaxed complex with the EcoRI require that the relaxation event take place at a unique site restriction endonuclease demonstrates that the single- in the DNA molecule. In this report, evidence will be pre- strand break is at a unique position. The site of the single- sented from studies using the EcoRI restriction endonuclease strand break in the relaxed ColEl complex is approximately the same distance from the EcoRI cleavage site as the that the strand-specific relaxation event takes place at a origin of ColEl DNA replication. unique site on both the ColEl and CoIE2 plasmid molecules. The location of this site, at least in the case of the ColEl Many of extrachromosomal circular DNA elements (plas- plasmid, is approximately the same distance from the single mids) of Escherichia coli, differing in molecular weight, num- EcoRI site as the origin of replication for this plasmid.
    [Show full text]
  • Topoisomerase II
    Topoisomerase II-␣ Expression in Different Cell Cycle Phases in Fresh Human Breast Carcinomas Kenneth Villman, M.D., Elisabeth Ståhl, M.S., Göran Liljegren, M.D., Ph.D., Ulf Tidefelt, M.D., Ph.D., Mats G. Karlsson, M.D., Ph.D. Departments of Oncology (KV), Pathology (ES, MGK), Surgery (GL), and Medicine (UT), Örebro University Hospital, Örebro, Sweden; and Karolinska Institute (UT), Stockholm, Sweden cluded in most adjuvant chemotherapy regimens Topoisomerase II-␣ (topo II␣) is the key target en- for breast cancer. Anthracyclines belong to the an- zyme for the topoisomerase inhibitor class of anti- ticancer agents called topoisomerase (topo) II cancer drugs. In normal cells, topo II␣ is expressed inhibitors. predominantly in the S/G2/M phase of the cell cycle. Topoisomerases are enzymes, present in the nu- In malignant cells, in vitro studies have indicated clei in mammalian cells, that regulate topological that the expression of topo II␣ is both higher and changes in DNA that are vital for many cellular less dependent on proliferation state in the cell. We processes such as replication and transcription (5). studied fresh specimens from 50 cases of primary They perform their function by introducing tran- breast cancer. The expression of topo II␣ in differ- sient protein-bridged DNA breaks on one (topo- ent cell cycle phases was analyzed with two- isomerase I) or both DNA strands (topoisomerase parameter flow cytometry using the monoclonal II; 6). There are two isoenzymes of topoisomerase antibody SWT3D1 and propidium iodide staining. II, with genetically and biochemical distinct fea- ␣ The expression of topo II was significantly higher tures.
    [Show full text]
  • The Obscure World of Integrative and Mobilizable Elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget
    The obscure world of integrative and mobilizable elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget To cite this version: Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget. The obscure world of integrative and mobilizable elements: Highly widespread elements that pirate bacterial conjugative systems. Genes, MDPI, 2017, 8 (11), pp.337. 10.3390/genes8110337. hal- 01686871 HAL Id: hal-01686871 https://hal.archives-ouvertes.fr/hal-01686871 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License G C A T T A C G G C A T genes Review The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements that Pirate Bacterial Conjugative Systems Gérard Guédon *, Virginie Libante, Charles Coluzzi, Sophie Payot and Nathalie Leblond-Bourget * ID DynAMic, Université de Lorraine, INRA, 54506 Vandœuvre-lès-Nancy, France; [email protected] (V.L.); [email protected] (C.C.); [email protected] (S.P.) * Correspondence: [email protected] (G.G.); [email protected] (N.L.-B.); Tel.: +33-037-274-5142 (G.G.); +33-037-274-5146 (N.L.-B.) Received: 12 October 2017; Accepted: 15 November 2017; Published: 22 November 2017 Abstract: Conjugation is a key mechanism of bacterial evolution that involves mobile genetic elements.
    [Show full text]
  • Virus World As an Evolutionary Network of Viruses and Capsidless Selfish Elements
    Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements Koonin, E. V., & Dolja, V. V. (2014). Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements. Microbiology and Molecular Biology Reviews, 78(2), 278-303. doi:10.1128/MMBR.00049-13 10.1128/MMBR.00049-13 American Society for Microbiology Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements Eugene V. Koonin,a Valerian V. Doljab National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, USAa; Department of Botany and Plant Pathology and Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon, USAb Downloaded from SUMMARY ..................................................................................................................................................278 INTRODUCTION ............................................................................................................................................278 PREVALENCE OF REPLICATION SYSTEM COMPONENTS COMPARED TO CAPSID PROTEINS AMONG VIRUS HALLMARK GENES.......................279 CLASSIFICATION OF VIRUSES BY REPLICATION-EXPRESSION STRATEGY: TYPICAL VIRUSES AND CAPSIDLESS FORMS ................................279 EVOLUTIONARY RELATIONSHIPS BETWEEN VIRUSES AND CAPSIDLESS VIRUS-LIKE GENETIC ELEMENTS ..............................................280 Capsidless Derivatives of Positive-Strand RNA Viruses....................................................................................................280
    [Show full text]
  • The Bacterial Conjugation Protein Trwb Resembles Ring Helicases And
    letters to nature metabolically labelled with BrdU (10 mM, 4 h), trypsinized, and ®xed with 70% ethanol. 17. Stampfer, M. R. et al. Gradual phenotypic conversion associated with immortalization of cultured Nuclei were isolated and stained with propidium iodide and FITC-conjugated anti-BrdU human mammary epithelial cells. Mol. Biol. Cell 8, 2391±2405 (1997). antibodies (Becton Dickinson, USA), as described7. Flow cytometry was performed on a 18. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. p53- and ATM-dependent apoptosis FACS Sorter (Becton Dickinson). All analysed events were gated to remove debris and induced by telomeres lacking TRF2. Science 283, 1321±1325 (1999). aggregates. 19. Artandi, S. E. et al. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice. Nature 406, 641±645 (2000). Cell death assays 20. Chin, L. et al. p53 De®ciency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell 97, 527±538 (1999). TUNEL assay for DNA fragmentation was done using an In Situ Cell Death Detection kit 21. Alcorta, D. A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4A) in replicative (BMB), according to manufacturer's protocol. Alternatively, cells were stained with senescence of normal human ®broblasts. Proc. Natl Acad. Sci. USA 92, 13742±13747 (1996). Annexin-V-FLUOR (BMB) and propidium iodide, and analysed by ¯uorescence 22. Hara, E. et al. Regulation of p16CDKN2 expression and its implications for cell immortalization and microscopy. senescence. Mol. Cell. Biol. 16, 859±867 (1996). 23. Burbano, R. R. et al.
    [Show full text]
  • RECQ5-Dependent Sumoylation of DNA Topoisomerase I Prevents Transcription-Associated Genome Instability
    ARTICLE Received 20 Aug 2014 | Accepted 23 Feb 2015 | Published 8 Apr 2015 DOI: 10.1038/ncomms7720 RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability Min Li1, Subhash Pokharel1,*, Jiin-Tarng Wang1,*, Xiaohua Xu1,* & Yilun Liu1 DNA topoisomerase I (TOP1) has an important role in maintaining DNA topology by relaxing supercoiled DNA. Here we show that the K391 and K436 residues of TOP1 are SUMOylated by the PIAS1–SRSF1 E3 ligase complex in the chromatin fraction containing active RNA polymerase II (RNAPIIo). This modification is necessary for the binding of TOP1 to RNAPIIo and for the recruitment of RNA splicing factors to the actively transcribed chromatin, thereby reducing the formation of R-loops that lead to genome instability. RECQ5 helicase promotes TOP1 SUMOylation by facilitating the interaction between PIAS1, SRSF1 and TOP1. Unexpectedly, the topoisomerase activity is compromised by K391/K436 SUMOylation, and this provides the first in vivo evidence that TOP1 activity is negatively regulated at transcriptionally active chromatin to prevent TOP1-induced DNA damage. Therefore, our data provide mechanistic insight into how TOP1 SUMOylation contributes to genome maintenance during transcription. 1 Department of Radiation Biology, Beckman Research Institute, City of Hope, Duarte, California 91010-3000, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.L. (email: [email protected]). NATURE COMMUNICATIONS | 6:6720 | DOI: 10.1038/ncomms7720 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7720 he prevention and efficient repair of DNA double-stranded transcriptionally active chromatin to prevent R-loops.
    [Show full text]
  • Sequence-Specific DNA Nicking Endonucleases
    BioMol Concepts 2015; 6(4): 253–267 Review Open Access Shuang-yong Xu* Sequence-specific DNA nicking endonucleases DOI 10.1515/bmc-2015-0016 Received May 20, 2015; accepted June 24, 2015 Introduction In this article, I will discuss natural DNA nicking endo- nucleases (NEases or nickases) with 3- to 7-bp specificities, Abstract: A group of small HNH nicking endonucleases e.g. Nt.CviPII (↓CCD, the down arrow indicates the nicked (NEases) was discovered recently from phage or prophage strand as shown) originally found in chlorella virus (1), genomes that nick double-stranded DNA sites ranging engineered NEases such as Nt.BspQI (GCTCTTCN↓), and from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site Nt.BbvCI (CC↓TCAGC) engineered from BspQI and BbvCI of phage HK97 contains a gp74 nicking site AC↑CGC, which restriction endonucleases (REases) (2, 3). The other group is similar to AC↑CGR (R = A/G) of N.φGamma encoded by of DNA NEases contains natural or engineered enzymes Bacillus phage Gamma. A minimal nicking domain of 76 with more than 8-bp target sites, which includes group I amino acid residues from N.φGamma could be fused to intron-encoded homing endonucleases (HEs) (4, 5), other DNA binding partners to generate chimeric NEases engineered nicking variants from LAGLIDAG HEs (6–8), with new specificities. The biological roles of a few small engineered TALE nucleases (TALENs) by fusion of tran- HNH endonucleases (HNHE, gp74 of HK97, gp37 of φSLT, scription activator-like effector (TALE) repeat domain with φ12 HNHE) have been demonstrated in phage and patho- FokI nuclease domain or a MutH nicking variant (9–11), ZF genicity island DNA packaging.
    [Show full text]
  • S Na P S H O T: D N a Mism a Tc H R E P a Ir
    SnapShot: Repair DNA Mismatch Scott A. Lujan, and Thomas Kunkel A. Larrea, Andres Park, NC 27709, USA Triangle Health Sciences, NIH, DHHS, Research National Institutes of Environmental 730 Cell 141, May 14, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.05.002 See online version for legend and references. SnapShot: DNA Mismatch Repair Andres A. Larrea, Scott A. Lujan, and Thomas A. Kunkel National Institutes of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA Mismatch Repair in Bacteria and Eukaryotes Mismatch repair in the bacterium Escherichia coli is initiated when a homodimer of MutS binds as an asymmetric clamp to DNA containing a variety of base-base and insertion-deletion mismatches. The MutL homodimer then couples MutS recognition to the signal that distinguishes between the template and nascent DNA strands. In E. coli, the lack of adenine methylation, catalyzed by the DNA adenine methyltransferase (Dam) in newly synthesized GATC sequences, allows E. coli MutH to cleave the nascent strand. The resulting nick is used for mismatch removal involving the UvrD helicase, single-strand DNA-binding protein (SSB), and excision by single-stranded DNA exonucleases from either direction, depending upon the polarity of the nick relative to the mismatch. DNA polymerase III correctly resynthesizes DNA and ligase completes repair. In bacteria lacking Dam/MutH, as in eukaryotes, the signal for strand discrimination is uncertain but may be the DNA ends associated with replication forks. In these bacteria, MutL harbors a nick-dependent endonuclease that creates a nick that can be used for mismatch excision. Eukaryotic mismatch repair is similar, although it involves several dif- ferent MutS and MutL homologs: MutSα (MSH2/MSH6) recognizes single base-base mismatches and 1–2 base insertion/deletions; MutSβ (MSH2/MSH3) recognizes insertion/ deletion mismatches containing two or more extra bases.
    [Show full text]