Spatial Organization and Dynamics of Rnase E and Ribosomes in Caulobacter Crescentus

Total Page:16

File Type:pdf, Size:1020Kb

Spatial Organization and Dynamics of Rnase E and Ribosomes in Caulobacter Crescentus Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus Camille A. Bayasa, Jiarui Wanga,b, Marissa K. Leea, Jared M. Schraderb,1, Lucy Shapirob,c, and W. E. Moernera,2 aDepartment of Chemistry, Stanford University, Stanford, CA 94305; bDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305; and cChan Zuckerberg Biohub, San Francisco, CA 94158 Contributed by W. E. Moerner, March 5, 2018 (sent for review December 15, 2017; reviewed by Zemer Gitai and James C. Weisshaar) We report the dynamic spatial organization of Caulobacter cres- components of the degradosome or the ribosome that are on centus RNase E (RNA degradosome) and ribosomal protein L1 (ri- length scales of less than ∼200 nm. bosome) using 3D single-particle tracking and superresolution We used a combination of live-cell single-particle tracking microscopy. RNase E formed clusters along the central axis of the (SPT) and fixed-cell superresolution (SR) microscopy to study the cell, while weak clusters of ribosomal protein L1 were deployed dynamics and spatial distribution of eYFP-labeled (13) RNase E throughout the cytoplasm. These results contrast with RNase E and ribosomal protein L1 in Caulobacter on subdiffraction limit and ribosome distribution in Escherichia coli, where RNase E coloc- length scales. SPT and SR provide improved resolution down to alizes with the cytoplasmic membrane and ribosomes accumulate in ∼20–50 nm using fluorescent proteins (FPs) (14, 15). Moreover, Cau- polar nucleoid-free zones. For both RNase E and ribosomes in both SPT and SR are single-molecule (SM) methods, allowing us lobacter , we observed a decrease in confinement and clustering to investigate the heterogeneity in protein behavior and distribu- upon transcription inhibition and subsequent depletion of nascent tion across many different cells. To avoid artifacts from projecting RNA, suggesting that RNA substrate availability for processing, deg- SM localizations onto two dimensions, we have used the double- radation, and translation facilitates confinement and clustering. Im- helix point spread function (DH-PSF), which encodes 3D infor- portantly, RNase E cluster positions correlated with the subcellular mation in each SM image (16). location of chromosomal loci of two highly transcribed rRNA genes, Using these methods, we observed that the number of RNase suggesting that RNase E’s function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome E clusters changed as a function of the cell cycle, while the size of and ribosome assembly are spatiotemporally organized in Caulo- the clusters remained constant. However, ribosomes formed only bacter, with chromosomal readout serving as the template for weak clusters, which were deployed throughout the cell, as we α this organization. found to be the case in another -bacterium, Sinorhizobium meliloti. For both RNase E and ribosomes, we observed a decrease ribosomes | RNA degradation | RNA processing | superresolution in confinement and clustering when transcription was inhibited, microscopy | single-molecule tracking likely due to depletion of RNA substrates for degradation, pro- cessing, and translation. We found that the localization of RNase E n bacteria, the RNA degradosome mediates the majority of clusters correlates with the subcellular chromosomal position of the ImRNA turnover and rRNA and tRNA maturation (1). The RNA degradosome assembles on the C-terminal scaffold region Significance of the RNase E endoribonuclease (2). RNase E in Escherichia coli and RNase Y, the RNase E homolog in Bacillus subtilis, both Regulated gene expression involves accurate subcellular posi- associate with the cell membrane through a membrane-binding tioning and timing of RNA synthesis, degradation, processing, helix (3–6). One proposed rationale behind the observed mem- and translation. RNase E is an endoribonuclease that forms the brane association is to physically separate transcription within scaffold of the RNA degradosome in bacteria, responsible for the nucleoid from RNA degradation, thus providing an inherent the majority of mRNA turnover and RNA processing. We used time delay between transcript synthesis and the onset of tran- 3D superresolution microscopy and single-particle tracking to script decay, avoiding a futile cycle. Caulobacter crescentus (Cau- quantify the spatial distribution and dynamics of RNase E and lobacter) is a Gram-negative α-proteobacterium widely studied as ribosomes in the asymmetrically dividing bacterium Caulo- a model system for asymmetric cell division. Unlike E. coli or B. bacter crescentus. Our results show that active transcription subtilis, Caulobacter contains a pole-tethered chromosome that and RNA substrate availability facilitate confinement and fills the cytoplasm (7–9). Surprisingly, Caulobacter RNase E does clustering of both RNase E and ribosomes. RNase E clusters not have a membrane-targeting sequence and is not membrane colocalized with the subcellular position of the two Caulo- associated (2, 10). Furthermore, diffraction-limited (DL) images bacter rRNA gene chromosomal loci, indicating that RNA pro- of RNase E exhibited a patchy localization throughout the cell, cessing can be spatially organized in a bacterium according to with RNase E directly or indirectly associating with DNA (10). its transcriptional profile. Moreover, the copy number of RNase E is regulated as a function of Author contributions: C.A.B., J.W., M.K.L., J.M.S., L.S., and W.E.M. designed research; the Caulobacter cell cycle. The copy number reaches maxima during C.A.B., J.W., and M.K.L. performed research; C.A.B., J.W., M.K.L., and J.M.S. contributed the swarmer to stalked cell transition and before cell division (2). new reagents/analytic tools; C.A.B., J.W., M.K.L., and W.E.M. analyzed data; and C.A.B., Fluorescently labeled ribosomal proteins S2 and L1, proxies J.W., L.S., and W.E.M. wrote the paper. for ribosomes in E. coli and B. subtilis, respectively, were found Reviewers: Z.G., Princeton University; and J.C.W., University of Wisconsin–Madison. enriched at the cell poles, spatially excluded from the nucleoid The authors declare no conflict of interest. on the hundreds of nanometer scale (11, 12). Similar fluores- Published under the PNAS license. cence imaging studies in Caulobacter revealed no apparent sep- 1Present address: Department of Biological Sciences, Wayne State University, Detroit, aration of ribosomes and the nucleoid, but did suggest that MI 48202. ribosome diffusion throughout the cell was spatially confined 2To whom correspondence should be addressed. Email: [email protected]. (10). However, because the Caulobacter cell is only ∼500 nm by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ∼3.5 μm in size, the DL resolution of conventional fluorescence 1073/pnas.1721648115/-/DCSupplemental. microscopy would obscure any organization or motion of the Published online April 2, 2018. E3712–E3721 | PNAS | vol. 115 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1721648115 Downloaded by guest on September 24, 2021 highly expressed rRNA genes that are positioned at two distinct and 0.1944 ± 0.0010 μm2/s (mobile). For rif-treated cells, the PNAS PLUS sites on the chromosome, consistent with processing occurring at diffusion coefficient for confined subtrajectories is 0.0060 ± the site of rRNA synthesis. Thus, cotranscriptional activities exhibit 0.0001 μm2/s and for mobile subtrajectories is 0.1270 ± dynamic subcellular organization that is dependent on the site of 0.0072 μm2/s. To determine whether the confined subtrajectories chromosomal readout. are stationary or not, the apparent diffusion coefficient of sta- tionary RNase E-eYFP molecules was calculated using the Results ensemble-averaged MSD of trajectories from fixed cells. Stationary, Diffusion of RNase E and Ribosomal L1 Proteins Is Influenced by fixed particles were found to have an apparent diffusion coefficient Transcriptional Activity. of 0.0022 ± 0.0015 μm2/s (error is SEM from 143 trajectories). RNase E. The endoribonuclease RNase E forms the core and Using this comparison, we determined that the observed diffusion scaffold of the Caulobacter RNA degradosome. Other compo- coefficient for WT confined subtrajectories is consistent with a nents include the exoribonuclease polynucleotide phosphorylase, stationary particle, whereas the diffusion coefficientforrifconfined a DEAD-box RNA helicase RhlB, the Krebs cycle enzyme aco- subtrajectories is larger than would be expected for a stationary nitase, and the exoribonuclease RNase D (2, 17). To study the particle given our localization precision (32/33/49 nm in x/y/z). motional dynamics of RNase E, we performed 3D SPT in a mixed The difference in diffusion between WT and rif-treated cells is population of live Caulobacter cells using a strain expressing not an artifact due to a potential rif-induced chromosome struc- RNase E-eYFP as the only copy of RNase E. We performed an ture disruption (22). We performed the same 3D SPT of RNase E initial photobleaching and waited for stochastic recovery and molecules in a ΔHU2 background with significantly reduced nu- blinking of eYFP SMs (18) (Fig. 1A, Left). We then obtained 3D cleoid compaction (23). Results show similar confinement as that trajectories of individual molecules by joining DH-PSF localiza- of WT cells (SI Appendix,Fig.S6), arguing against the possibility tions over consecutive frames (Materials and Methods) (Fig. 1A, of a difference in chromosome packaging between WT and rif- Right). In contrast to E. coli in which RNase E diffuses on the treated cells contributing to the difference in RNase E diffusion. membrane (3), RNase E molecules in Caulobacter (average copy Ribosomes. To obtain information about ribosome dynamics and number is 3,100 molecules per cell as determined for a mixed cell translation, we performed 3D SPT of ribosomes through imaging population; SI Appendix, Supplementary Methods)werefound eYFP-labeled ribosomal protein L1 in a mixed population of throughout the cytoplasm of the cell, diffusing limited distances cells. We found that L1 (average copy number is 16,300 mole- (Fig.
Recommended publications
  • Telomeres.Pdf
    Telomeres Secondary article Elizabeth H Blackburn, University of California, San Francisco, California, USA Article Contents . Introduction Telomeres are specialized DNA–protein structures that occur at the ends of eukaryotic . The Replication Paradox chromosomes. A special ribonucleoprotein enzyme called telomerase is required for the . Structure of Telomeres synthesis and maintenance of telomeric DNA. Synthesis of Telomeric DNA by Telomerase . Functions of Telomeres Introduction . Telomere Homeostasis . Alternatives to Telomerase-generated Telomeric DNA Telomeres are the specialized chromosomal DNA–protein . Evolution of Telomeres and Telomerase structures that comprise the terminal regions of eukaryotic chromosomes. As discovered through studies of maize and somes. One critical part of this protective function is to fruitfly chromosomes in the 1930s, they are required to provide a means by which the linear chromosomal DNA protect and stabilize the genetic material carried by can be replicated completely, without the loss of terminal eukaryotic chromosomes. Telomeres are dynamic struc- DNA nucleotides from the 5’ end of each strand of this tures, with their terminal DNA being constantly built up DNA. This is necessary to prevent progressive loss of and degraded as dividing cells replicate their chromo- terminal DNA sequences in successive cycles of chromo- somes. One strand of the telomeric DNA is synthesized by somal replication. a specialized ribonucleoprotein reverse transcriptase called telomerase. Telomerase is required for both
    [Show full text]
  • Replication Stress: an Achilles' Heel of Glioma Cancer Stem–Like Cells Meredith A
    Published OnlineFirst November 29, 2018; DOI: 10.1158/0008-5472.CAN-18-2439 Cancer Review Research Replication Stress: An Achilles' Heel of Glioma Cancer Stem–like Cells Meredith A. Morgan1, and Christine E. Canman2 Abstract Glioblastoma (GBM) is a highly aggressive form of cancer that Carruthers and colleagues investigated DNA replication stress as is resistant to standard therapy with concurrent radiation and an underlying mechanism responsible for upregulation of the temozolomide, two agents that work by inducing DNA damage. DDR and hence the radiation resistance of glioma stem–like An underlying causeof this resistance may be a subpopulation of cells. Furthermore, the authors explore the efficacy of combined cancer stem–like cells that display a heightened DNA damage ataxia telangiectasia and Rad3-related kinase and PARP inhibi- response (DDR). Although this DDR represents an attractive tors as a strategy to leverage these mechanisms and overcome therapeutic target for overcoming the resistance of GBMs to radiation resistance. Cancer Res; 78(24); 6713–6. Ó2018 AACR. radiotherapy, until now, the cause of this DDR upregulation has See related article by Carruthers and colleagues, Cancer Res; not been understood. In a previous issue of Cancer Research, 78(17); 5060–71. The cancer stem cell theory states that a small subpopulation of of replication factories compared with non-GSC populations tumor cells possess unique self-renewal properties that are capa- (6). These observations pointed to elevated levels of RS as ble of seeding new tumors and are a source of regrowth following causative of DDR activation in untreated GSCs, a hypothesis therapy (1). Glioma stem–like cells (GSC) are defined as CD133- supported by the high levels of RS in glioblastoma (GBM; positive cells that can initiate new tumors in mice (2).
    [Show full text]
  • Congenital Microcephaly
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Sussex Research Online American Journal of Medical Genetics Part C (Seminars in Medical Genetics) ARTICLE Congenital Microcephaly DIANA ALCANTARA AND MARK O'DRISCOLL* The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature. © 2014 Wiley Periodicals, Inc. KEY WORDS: cell division; mitosis; DNA replication; cilia How to cite this article: Alcantara D, O'Driscoll M. 2014. Congenital microcephaly. Am J Med Genet Part C Semin Med Genet 9999:1–16. INTRODUCTION mid‐gestation although glial cell division formation of the various cortical layers. and consequent brain volume enlarge- Furthermore, differentiating and devel- Congenital microcephaly, an occipital‐ ment does continue after birth [Spalding oping neurons must migrate to their frontal circumference of equal to or less et al., 2005]. Impaired neurogenesis is defined locations to construct the com- than 2–3 standard deviations below the therefore most obviously reflected clini- plex architecture and laminar layered age‐related population mean, denotes cally as congenital microcephaly.
    [Show full text]
  • POLD3 Is Haploinsufficient for DNA Replication in Mice
    POLD3 is haploinsufficient for DNA replication in mice Matilde Murga1, Emilio Lecona1, Irene Kamileri2, Marcos Díaz3, Natalia Lugli2, Sotirios K. Sotiriou2, Marta E. Anton1, Juan Méndez3, Thanos D. Halazonetis2 and Oscar Fernandez-Capetillo1,4 1 Genomic Instability Group, Spanish National Cancer Research Centre, Madrid, Spain. 2Department of Molecular Biology, University of Geneva, Geneva, Switzerland. 3 DNA Replication Group, Spanish National Cancer Research Centre, Madrid, Spain. 4Science for Life Laboratories, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. Correspondence: O.F. ([email protected]) Contact: Oscar Fernandez-Capetillo Spanish National Cancer Research Centre (CNIO) Melchor Fernandez Almagro, 3 Madrid 28029, Spain Tel.: +34.91.732.8000 Ext: 3480 Fax: +34.91.732.8028 Email: [email protected] POLD3 deficient mice SUMMARY The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologues are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizzosaccharomyces pombe orthologue is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3+/- mice were born at sub-Mendelian ratios and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.
    [Show full text]
  • DNA Polymerase Δ Proofreads Errors Made by DNA Polymerase E
    DNA polymerase δ proofreads errors made by DNA polymerase e Chelsea R. Bulocka, Xuanxuan Xinga,1, and Polina V. Shcherbakovaa,2 aEppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198 Edited by Sue Jinks-Robertson, Duke University School of Medicine, Durham, NC, and approved February 5, 2020 (received for review October 9, 2019) − During eukaryotic replication, DNA polymerases e (Pole) and δ analogous Pole-exo variant (2, 17–25). Furthermore, haploid (Polδ) synthesize the leading and lagging strands, respectively. In yeast deficient in Polδ proofreading do not survive when DNA a long-known contradiction to this model, defects in the fidelity of mismatch repair (MMR) is also inactivated with the death at- Pole have a much weaker impact on mutagenesis than analogous tributed to an excessive level of mutagenesis (26). In contrast, yeast Polδ defects. It has been previously proposed that Polδ contributes lacking both proofreading by Pole and MMR are viable, and while more to mutation avoidance because it proofreads mismatches the mutation rate in these strains is high, it does not reach the created by Pole in addition to its own errors. However, direct lethal threshold (19, 21, 22, 25, 27). Similarly, when identical ty- evidence for this model was missing. We show that, in yeast, rosine to alanine substitutions were made in the conserved region the mutation rate increases synergistically when a Pole nucleotide III of the polymerase domains (Polδ-Y708A and Pole-Y831A), the selectivity defect is combined with a Polδ proofreading defect, dem- Polδ variant produced a much stronger mutator effect than the onstrating extrinsic proofreading of Pole errors by Polδ.
    [Show full text]
  • Functional Characterization of the DNA Polymerase Epsilon and Its Involvement in the Maintenance of Genome Integrity in Arabidopsis Jose Antonio Pedroza-Garcia
    Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Jose Antonio Pedroza-Garcia To cite this version: Jose Antonio Pedroza-Garcia. Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis. Plants genetics. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS248. tel-03092326 HAL Id: tel-03092326 https://tel.archives-ouvertes.fr/tel-03092326 Submitted on 2 Jan 2021 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. NNT : 2016SACLS248 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY, préparée à l’Université Paris-Sud ÉCOLE DOCTORALE N° 567 Sciences du Végétal : du Gène à l’Ecosystème Spécialité de doctorat: Biologie Par M. José Antonio Pedroza-Garcia Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Thèse présentée et soutenue à Orsay, le 22 septembre 2016 : Composition du Jury : M. Frugier, Florian Directeur de Recherche, CNRS Président Mme Gallego, Maria Professeure, Université Blaise Pascal Rapporteur M. Bendahmane, Mohammed Directeur de Recherche, INRA Rapporteur Mme Mézard, Christine Directrice de Recherche, CNRS Examinatrice Mme Chabouté, Marie-Edith Directrice de Recherche, CNRS Examinatrice Mme Raynaud, Cécile Chargée de Recherche, CNRS Directrice de thèse Acknowledgments First, I would like to express my huge gratitude to my supervisor, Cécile Raynaud.
    [Show full text]
  • Ribonuclease E Organizes the Protein Interactions in the Escherichia Coli RNA Degradosome
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome Nathalie F. Vanzo,1 Yeun Shan Li,2 Be´atrice Py,2,3 Erwin Blum,2 Christopher F. Higgins,2,4 Lelia C. Raynal,1 Henry M. Krisch,1 and Agamemnon J. Carpousis1,5 1Laboratoire de Microbiologie et Ge´ne´tique Mole´culaire, UPR 9007, Centre National de la Recherche Scientifique (CNRS), 31062 Toulouse Cedex, France; 2Nuffield Department of Clinical Biochemistry and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented.
    [Show full text]
  • The Structure and Function of the Gram-Positive Bacterial RNA Degradosome
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector REVIEW published: 03 February 2017 doi: 10.3389/fmicb.2017.00154 The Structure and Function of the Gram-Positive Bacterial RNA Degradosome Kyu Hong Cho* Department of Biology, Indiana State University, Terre Haute, IN, USA The RNA degradosome is a highly structured protein complex responsible for bulk RNA decay in bacteria. The main components of the complex, ribonucleases, an RNA helicase, and glycolytic enzymes are well-conserved in bacteria. Some components of the degradosome are essential for growth and the disruption of degradosome formation causes slower growth, indicating that this complex is required for proper cellular function. The study of the Escherichia coli degradosome has been performed extensively for the last several decades and has revealed detailed information on its structure and function. On the contrary, the Gram-positive bacterial degradosome, which contains ribonucleases different from the E. coli one, has been studied only recently. Studies on the Gram-positive degradosome revealed that its major component RNase Y was necessary for the full virulence of medically important Gram-positive bacterial pathogens, Edited by: suggesting that it could be a target of antimicrobial therapy. This review describes the Marc Bramkamp, Ludwig Maximilian University of structures and function of Gram-positive bacterial RNA degradosomes, especially those Munich, Germany of a Gram-positive model organism Bacillus subtilis, and two important Gram-positive Reviewed by: pathogens, Staphylococcus aureus and Streptococcus pyogenes. Jörg Stülke, University of Göttingen, Germany Keywords: Gram-positive RNA degradosome, B. subtilis, S.
    [Show full text]
  • Lecture 1 Enzymes in Genetic Engineering: Restriction Nucleases
    NPTEL – Bio Technology – Genetic Engineering & Applications MODULE 2: LECTURE 1 ENZYMES IN GENETIC ENGINEERING: RESTRICTION NUCLEASES: EXO & ENDO NUCLEASES 2-1.1 Introduction: A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as restriction sites. In bacteria, restriction enzymes form a combined system (restriction + modification system) with modification enzymes that methylate the bacterial DNA. Methylation of bacterial DNA at the recognition sequence typically protects the own DNA of the bacteria from being cleaved by restriction enzyme. There are two different kinds of restriction enzymes: (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II etc. (2) Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc. 2-1.2History: In 1970 the first restriction endonuclease enzyme HindII was isolated. For the subsequent discovery and characterization of numerous restriction endonucleases, in 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for Physiology or Medicine. Since then, restriction enzymes have been used as an essential tool in recombinant DNA technology. Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 30 NPTEL – Bio Technology – Genetic Engineering & Applications 2-1.3 Restriction Endonuclease Nomenclature: Restriction endonucleases are named according to the organism in which they were discovered, using a system of letters and numbers. For example, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d).
    [Show full text]
  • Evidence for a Degradosome-Like Complex in the Mitochondria of Trypanosoma Brucei
    FEBS Letters 583 (2009) 2333–2338 journal homepage: www.FEBSLetters.org Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei Jonelle L. Mattiacio 1, Laurie K. Read * Depatment of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY 14214, USA article info abstract Article history: Mitochondrial RNA turnover in yeast involves the degradosome, composed of DSS-1 exoribonuc- Received 29 May 2009 lease and SUV3 RNA helicase. Here, we describe a degradosome-like complex, containing SUV3 Accepted 11 June 2009 and DSS-1 homologues, in the early branching protozoan, Trypanosoma brucei. TbSUV3 is mitoc- Available online 18 June 2009 hondrially localized and co-sediments with TbDSS-1 on glycerol gradients. Co-immunoprecipitation demonstrates that TbSUV3 and TbDSS-1 associate in a stable complex, which differs from the yeast Edited by Michael Ibba degradosome in that it is not stably associated with mitochondrial ribosomes. This is the first report of a mitochondrial degradosome-like complex outside of yeast. Our data indicate an early evolution- Keywords: ary origin for the mitochondrial SUV3/DSS-1 containing complex. RNA turnover RNA processing Trypanosome Structured summary: RNA helicase MINT-7187980: SUV3 (genbank_protein_gi:XP_844349) and DSS1 (uniprotkb:Q38EM3) colocalize Exoribonuclease (MI:0403) by cosedimentation (MI:0027) MINT-7188074: SUV3 (genbank_protein_gi:XP_844349) physically interacts (MI:0914) with DSS1 (uni- protkb:Q38EM3) by anti tag co-immunoprecipitation (MI:0007) Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction only known exoribonuclease involved in yeast mitochondrial RNA (mtRNA) turnover [8].
    [Show full text]
  • CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells
    Author Manuscript Published OnlineFirst on March 11, 2020; DOI: 10.1158/0008-5472.CAN-19-1372 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: CHK1 inhibition is synthetically lethal with loss of B-family DNA polymerase function in human lung and colorectal cancer cells. Author List: Rebecca F. Rogers1, Mike I. Walton1, Daniel L. Cherry2, Ian Collins1, Paul A. Clarke1, Michelle D. Garrett2* and Paul Workman1* Affiliations: 1Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, SM2 5NG, UK 2 School of Biosciences, Stacey Building, University of Kent, Canterbury, Kent, CT2 7NJ, UK Running title: Synthetic lethality of CHK1 and DNA polymerase inhibition *Corresponding authors: Michelle D Garrett and Paul Workman Corresponding Author Information Michelle D Garrett School of Biosciences, Stacey Building, University of Kent, Canterbury, Kent, CT2 7NJ, UK. Email: [email protected] Phone: +44 (0)1227 816140 Paul Workman Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, SM2 5NG, UK. Email: [email protected] Phone: +44 (0)20 7153 5209 Conflict of interest statement: IC, MDG, MIW, RFR, PC and PW are current or past employees of The Institute of Cancer Research, which has a commercial interest in the discovery and development of CHK1 inhibitors, including SRA737, and operates a rewards-to- inventors scheme. IC, MDG and MIW have been involved in a commercial collaboration on CHK1 inhibitors with Sareum Ltd and intellectual property arising from the program, including SRA737, was licensed to Sierra Oncology. IC is a consultant for Sierra Oncology, Adorx Ltd, Epidarex LLP and Enterprise Therapeutics Ltd and holds equity in Monte Rosa Therapeutics AG.
    [Show full text]
  • DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer
    cancers Review DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer Youri I. Pavlov 1,2,* , Anna S. Zhuk 3 and Elena I. Stepchenkova 2,4 1 Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Genetics and Biotechnology, Saint-Petersburg State University, 199034 Saint Petersburg, Russia; [email protected] 3 International Laboratory of Computer Technologies, ITMO University, 197101 Saint Petersburg, Russia; [email protected] 4 Laboratory of Mutagenesis and Genetic Toxicology, Vavilov Institute of General Genetics, Saint-Petersburg Branch, Russian Academy of Sciences, 199034 Saint Petersburg, Russia * Correspondence: [email protected] Received: 30 September 2020; Accepted: 13 November 2020; Published: 24 November 2020 Simple Summary: The etiology of cancer is linked to the occurrence of mutations during the reduplication of genetic material. Mutations leading to low replication fidelity are the culprits of many hereditary and sporadic cancers. The archetype of the current model of replication fork was proposed 30 years ago. In the sequel to our 2010 review with the words “years after” in the title inspired by A. Dumas’s novels, we go over new developments in the DNA replication field and analyze how they help elucidate the effects of the genetic variants of DNA polymerases on cancer. Abstract: Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization.
    [Show full text]