Dna Replication (Part – Iii)
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Mutations That Separate the Functions of the Proofreading Subunit of the Escherichia Coli Replicase
G3: Genes|Genomes|Genetics Early Online, published on April 15, 2015 as doi:10.1534/g3.115.017285 Mutations that separate the functions of the proofreading subunit of the Escherichia coli replicase Zakiya Whatley*,1 and Kenneth N Kreuzer*§ *University Program in Genetics & Genomics, Duke University, Durham, NC 27705 §Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 1 © The Author(s) 2013. Published by the Genetics Society of America. Running title: E. coli dnaQ separation of function mutants Keywords: DNA polymerase, epsilon subunit, linker‐scanning mutagenesis, mutation rate, SOS response Corresponding author: Kenneth N Kreuzer, Department of Biochemistry, Box 3711, Nanaline Duke Building, Research Drive, Duke University Medical Center, Durham, NC 27710 Phone: 919 684 6466 FAX: 919 684 6525 Email: [email protected] 1 Present address: Department of Biology, 300 N Washington Street, McCreary Hall, Campus Box 392, Gettysburg College, Gettysburg, PA 17325 Phone: 717 337 6160 Fax: 7171 337 6157 Email: [email protected] 2 ABSTRACT The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3’ 5’ exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our lab identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response following quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. -
Structure of the Human Clamp Loader Reveals an Autoinhibited Conformation of a Substrate-Bound AAA+ Switch
Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch Christl Gaubitza,1, Xingchen Liua,b,1, Joseph Magrinoa,b, Nicholas P. Stonea, Jacob Landecka,b, Mark Hedglinc, and Brian A. Kelcha,2 aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester MA 01605; bGraduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester MA 01605; and cDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802 Edited by Michael E. O’Donnell, HHMI and Rockefeller University, New York, NY, and approved July 27, 2020 (received for review April 20, 2020) DNA replication requires the sliding clamp, a ring-shaped protein areflexia syndrome (15), Hutchinson–Gilford progeria syn- complex that encircles DNA, where it acts as an essential cofactor drome (16), and in the replication of some viruses (17–19). It for DNA polymerases and other proteins. The sliding clamp needs is unknown whether loading by RFC contributes to PARD to be opened and installed onto DNA by a clamp loader ATPase of disease. the AAA+ family. The human clamp loader replication factor C Clamp loaders are members of the AAA+ family of ATPases (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) (ATPases associated with various cellular activities), a large are both essential and play critical roles in several diseases. De- protein family that uses the chemical energy of adenosine 5′- spite decades of study, no structure of human RFC has been re- triphosphate (ATP) to generate mechanical force (20). Most solved. Here, we report the structure of human RFC bound to AAA+ proteins form hexameric motors that use an undulating PCNA by cryogenic electron microscopy to an overall resolution ∼ spiral staircase mechanism to processively translocate a substrate of 3.4 Å. -
DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine
Annu. Rev. Biochem. 1995.64:171-200 Copyright Ii) 1995 byAnnual Reviews Inc. All rights reserved DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine Zvi Kelman and Mike O'Donnell} Microbiology Department and Hearst Research Foundation. Cornell University Medical College. 1300York Avenue. New York. NY }0021 KEY WORDS: DNA replication. multis ubuni t complexes. protein-DNA interaction. DNA-de penden t ATPase . DNA sliding clamps CONTENTS INTRODUCTION........................................................ 172 THE HOLO EN ZYM E PARTICL E. .......................................... 173 THE CORE POLYMERASE ............................................... 175 THE � DNA SLIDING CLAM P............... ... ......... .................. 176 THE yC OMPLEX MATCHMAKER......................................... 179 Role of ATP . .... .............. ...... ......... ..... ............ ... 179 Interaction of y Complex with SSB Protein .................. ............... 181 Meclwnism of the yComplex Clamp Loader ................................ 181 Access provided by Rockefeller University on 08/07/15. For personal use only. THE 't SUBUNIT . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 182 Annu. Rev. Biochem. 1995.64:171-200. Downloaded from www.annualreviews.org AS YMMETRIC STRUC TURE OF HOLO EN ZYM E . 182 DNA PO LYM ER AS E III HOLO ENZ YME AS A REPLIC ATING MACHINE ....... 186 Exclwnge of � from yComplex to Core .................................... 186 Cycling of Holoenzyme on the LaggingStrand -
Restoration of Mir-193A Expression Is Tumor-Suppressive in MYC
Bharambe et al. Acta Neuropathologica Communications (2020) 8:70 https://doi.org/10.1186/s40478-020-00942-5 RESEARCH Open Access Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma Harish Shrikrishna Bharambe1,2, Annada Joshi1, Kedar Yogi1,2, Sadaf Kazi1 and Neelam Vishwanath Shirsat1,2* Abstract Medulloblastoma, a highly malignant pediatric brain tumor, consists of four molecular subgroups, namely WNT, SHH, Group 3, and Group 4. The expression of miR-193a, a WNT subgroup-specific microRNA, was found to be induced by MYC, an oncogenic target of the canonical WNT signaling. MiR-193a is not expressed in Group 3 medulloblastomas, despite MYC expression, as a result of promoter hypermethylation. Restoration of miR-193a expression in the MYC amplified Group 3 medulloblastoma cells resulted in inhibition of growth, tumorigenicity, and an increase in radiation sensitivity. MAX,STMN1, and DCAF7 were identified as novel targets of miR-193a. MiR- 193a mediated downregulation of MAX could suppress MYC activity since it is an obligate hetero-dimerization partner of MYC. MYC induced expression of miR-193a, therefore, seems to act as a feedback inhibitor of MYC signaling. The expression of miR-193a resulted in widespread repression of gene expression that included not only several cell cycle regulators, WNT, NOTCH signaling genes, and those encoding DNA replication machinery, but also several chromatin modifiers like SWI/SNF family genes and histone-encoding genes. MiR-193a expression brought about a reduction in the global levels of H3K4me3, H3K27ac, the histone marks of active chromatin, and an increase in the levels of H3K27me3, a repressive chromatin mark. -
Rnase-H-Based Assays Utilizing Modified Rna
(19) TZZ ¥_T (11) EP 2 279 263 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) 04.09.2013 Bulletin 2013/36 (86) International application number: (21) Application number: 09739895.2 PCT/US2009/042454 (22) Date of filing: 30.04.2009 (87) International publication number: WO 2009/135093 (05.11.2009 Gazette 2009/45) (54) RNASE-H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS TESTS AUF RNASE-H-BASIS MIT MODIFIZIERTEN RNA-MONOMEREN DOSAGES À BASE DE RNASE-H UTILISANT DES MONOMÈRES D’ARN MODIFIÉS (84) Designated Contracting States: • ROSE, Scott AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Coralville, IA 52241 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL • DOBOSY, Joseph PT RO SE SI SK TR Coralville, IA 52241 (US) (30) Priority: 30.04.2008 US 49204 P (74) Representative: Grünecker, Kinkeldey, Stockmair & Schwanhäusser (43) Date of publication of application: Leopoldstrasse 4 02.02.2011 Bulletin 2011/05 80802 München (DE) (60) Divisional application: (56) References cited: 13173388.3 EP-A- 1 367 136 WO-A-01/21813 13173389.1 WO-A-03/074724 WO-A-2004/059012 13173390.9 WO-A-2007/062495 WO-A2-2005/021776 13173391.7 WO-A2-2007/141580 US-A- 5 744 308 US-A- 5 830 664 (73) Proprietor: Integrated Dna Technologies, Inc. Coralville, IA 52241 (US) • ITAYA M ET AL: "Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme (72) Inventors: thermophile Thermus thermophilus HB8: a • WALDER, Joseph, Alan thermostable RNase H can functionally replace Chicago, IL 60645 (US) the Escherichia coli enzyme in vivo." NUCLEIC • BEHLKE, Mark, Aaron ACIDS RESEARCH 25 AUG 1991, vol. -
DNA Replication
Predicted by Watson & Crick model CONSERVATIVE (one totally new, one totally old) Other possibilities DISPERSIVE (each have mixed bits) Grow E-Coli in 15N for several generation [15NH4Cl as sole N-source) Transfer to medium containing only 14N Extract DNA after each replication Semi-conservative ISOPYCNIC density gradient centrifugation Components stop at the point in the Measure density using CsCl gradient gradient equal to their buoyant density Experiment In the second generation, ALL DNA has a density halfway in between 15N DNA and 14N DNA In subsequent generations, the proportion of fully 14N DNA increases, but some hybrids remain DNA polymerases synthesise DNA in the 5' > 3' direction [because the 3' OH attacks the incoming nucleotide] DNA is antiparallel Both new strands are synthesized simultaneously at the replication fork If DNA replication is semi-conservative, this poses a problem Bacterial chromosomes contain a SINGLE Therefore, one of the strands needs to grow ORIGIN, bound to the cell membrane, in the 3' > 5' direction within the oriC locus Enter Sub-topic Contains four 9 bp binding sites for the Synthesis of this protein is coupled to growth rate initiator protein DnaA Add a pulse of 3H-thymidine to cells Quench and harvest DNA Once it has attained a critical level, DnaA forms a complex of 30-40 molecules, each Requires DNA to be NEGATIVELY bound to ATP, around which oriC DNA Experiment 1 SUPERCOILED becomes wrapped Initiation Facilitates the MELTING of three 13 bp Separate by size AT-rich repeat sequences, which open toallow binding of DnaB (DNA helicase) Add a pulse of 3H-thymidine to cells DNA primase then binds and synthesizes a "Chase" with unlabelled thymidine short RNA primer on the LEADING DNA polymerases and STRAND. -
Telomerase Reverse Transcriptase Expression Elevated by Avian Leukosis Virus Integration in B Cell Lymphomas
Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas Feng Yang, Rena R. Xian*, Yingying Li, Tatjana S. Polony, and Karen L. Beemon† Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 Communicated by Saul Roseman, The Johns Hopkins University, Baltimore, MD, September 26, 2007 (received for review May 11, 2007) Simple retroviruses induce tumors by integrating into the host integration sites are believed to result from strong biological genome, activating cellular oncogenes and microRNAs, or inacti- selection (7). vating tumor suppressor genes. The identification of these genes Avian leukosis virus (ALV) is a simple retrovirus that does not elucidates molecular mechanisms of tumorigenesis. In this study, encode an oncogene but that can induce tumors by integrating we identified avian leukosis virus (ALV) proviral integration sites in near oncogenes, introducing a strong promoter or enhancer rapid-onset B cell lymphomas arising <12 weeks after infection of sequence (8–12). Typically, ALV induces B cell lymphomas to chicken embryos. By using inverse PCR, 28 unique viral integration develop in Ϸ6 months and frequently involves proviral integra- sites were identified in rapid-onset tumors. Integrations in the tions, resulting in deregulation of the cellular transcription telomerase reverse transcriptase (TERT) promoter/enhancer region factor, myc, as well as bic (precursor to microRNA 155) (10–12). were observed in four different tumors, suggesting that this is a In addition, B cell lymphomas with a more rapid onset (lethal in common integration site. These provirus integrations ranged from Ͻ3 months) have been observed with clonal proviral insertions 217 to 2,584 bp upstream of the TERT transcription initiation site into the myb gene locus, resulting in overexpression of a trun- and were all in the opposite transcriptional orientation to TERT. -
Hexameric Helicase G40P Unwinds DNA in Single Base Pair Steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7*
RESEARCH ARTICLE Hexameric helicase G40P unwinds DNA in single base pair steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7* 1Physics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States; 2B CUBE – Center for Molecular Bioengineering, Technische Universita¨ t Dresden, Dresden, Germany; 3Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States; 4Howard Hughes Medical Institute, Baltimore, United States; 5Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States; 6Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States; 7Department of Biophysics, Johns Hopkins University, Baltimore, United States Abstract Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPgS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis. -
The Replisome Guides Nucleosome Assembly During DNA Replication Wenshuo Zhang, Jianxun Feng and Qing Li*
Zhang et al. Cell Biosci (2020) 10:37 https://doi.org/10.1186/s13578-020-00398-z Cell & Bioscience REVIEW Open Access The replisome guides nucleosome assembly during DNA replication Wenshuo Zhang, Jianxun Feng and Qing Li* Abstract Nucleosome assembly during DNA replication is tightly coupled to ongoing DNA synthesis. This process, termed DNA replication-coupled (RC) nucleosome assembly, is essential for chromatin replication and has a great impact on both genome stability maintenance and epigenetic inheritance. This review discusses a set of recent fndings regarding the role of replisome components contributing to RC nucleosome assembly. Starting with a brief introduction to the fac- tors involved in nucleosome assembly and some aspects of the architecture of the eukaryotic replisome, we discuss studies from yeast to mammalian cells and the interactions of replisome components with histones and histone chaperones. We describe the proposed functions of replisome components during RC nucleosome assembly and discuss their impacts on histone segregation and implications for epigenetic inheritance. Keywords: Replisome component, Nucleosome assembly, Chromatin replication, Histone chaperone Background state. Tis process, called DNA replication-coupled (RC) A brief introduction to DNA replication‑coupled (RC) nucleosome assembly, is an essential step for chromatin nucleosome assembly replication [2, 4, 6]. Eukaryotic DNA replication occurs in the context of Nucleosome assembly during DNA replication occurs the chromatin environment [1]. Chromatin, the carrier in a stepwise fashion. Early studies using a chemical of genetic and epigenetic information and guardian of cross-linking technique combined with radioisotope genome stability, must be duplicated in daughter cells labeling methods demonstrated that parental histone to ensure continuity between generations. -
Telomere Maintenance Pathway Activity Analysis Enables Tissue- and Gene-Level Inferences
bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429081; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Telomere maintenance pathway activity analysis enables tissue- and gene-level inferences Lilit Nersisyan1,2*, Arman Simonyan1, Hans Binder3, Arsen Arakelyan1,2 1 Bioinformatics Group, Institute of Molecular Biology, National Academy of Sciences, Yerevan, Armenia 2 Pathverse, LLC, Yerevan, Armenia 3 Interdisciplinary Center for Bioinformatics, University of Leipzig, Leipzig, Germany * Correspondence: Lilit Nersisyan Keywords: telomere maintenance mechanisms, telomerase, alternative lengthening of telomeres, pathway signal flow, testis ABSTRACT Telomere maintenance is one of the mechanisms ensuring indefinite divisions of cancer and stem cells. Good understanding of telomere maintenance mechanisms (TMM) is important for studying cancers and designing therapies. However, molecular factors triggering selective activation of either the telomerase dependent (TEL) or the alternative lengthening of telomeres (ALT) pathway are poorly understood. In addition, more accurate and easy-to-use methodologies are required for TMM phenotyping. In this study, we have performed literature based reconstruction of signaling pathways for the ALT and TEL TMMs. Gene expression data were used for computational assessment of TMM pathway activities and compared with experimental assays for TEL and ALT. Explicit consideration of pathway topology makes bioinformatics analysis more informative compared to computational methods based on simple summary measures of gene expression. Application to healthy human tissues showed high ALT and TEL pathway activities in testis, and identified genes and pathways that may trigger TMM activation. -
Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms
International Journal of Molecular Sciences Review Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms Megan Schmit and Anja-Katrin Bielinsky * Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] * Correspondence: [email protected] Abstract: Deoxyribonucleic acid (DNA) replication can be divided into three major steps: initiation, elongation and termination. Each time a human cell divides, these steps must be reiteratively carried out. Disruption of DNA replication can lead to genomic instability, with the accumulation of point mutations or larger chromosomal anomalies such as rearrangements. While cancer is the most common class of disease associated with genomic instability, several congenital diseases with dysfunctional DNA replication give rise to similar DNA alterations. In this review, we discuss all congenital diseases that arise from pathogenic variants in essential replication genes across the spectrum of aberrant replisome assembly, origin activation and DNA synthesis. For each of these conditions, we describe their clinical phenotypes as well as molecular studies aimed at determining the functional mechanisms of disease, including the assessment of genomic stability. By comparing and contrasting these diseases, we hope to illuminate how the disruption of DNA replication at distinct steps affects human health in a surprisingly cell-type-specific manner. Keywords: Meier-Gorlin syndrome; natural killer cell deficiency; X-linked pigmentary reticulate disorder; Van Esch-O’Driscoll disease; IMAGe syndrome; FILS syndrome; Rothmund-Thomson syndrome; Baller-Gerold syndrome; RAPADILINO Citation: Schmit, M.; Bielinsky, A.-K. Congenital Diseases of DNA Replication: Clinical Phenotypes and 1. Introduction Molecular Mechanisms. Int. J. Mol. 1.1. Replication Initiation Sci. -
Potential of Naturally Derived Compounds in Telomerase and Telomere Modulation in Skin Senescence and Aging
International Journal of Molecular Sciences Review Potential of Naturally Derived Compounds in Telomerase and Telomere Modulation in Skin Senescence and Aging Barbara Jacczak, Błazej˙ Rubi´s and Ewa Toto´n* Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, 49 Przybyszewskiego St., 60-355 Pozna´n,Poland; [email protected] (B.J.); [email protected] (B.R.) * Correspondence: [email protected]; Tel.: +48-61-869-1427 Abstract: Proper functioning of cells—their ability to divide, differentiate, and regenerate—is dictated by genomic stability. The main factors contributing to this stability are the telomeric ends that cap chromosomes. Telomere biology and telomerase activity have been of interest to scientists in various medical science fields for years, including the study of both cancer and of senescence and aging. All these processes are accompanied by telomere-length modulation. Maintaining the key levels of telomerase component (hTERT) expression and telomerase activity that provide optimal telomere length as well as some nontelomeric functions represents a promising step in advanced anti-aging strategies, especially in dermocosmetics. Some known naturally derived compounds contribute significantly to telomere and telomerase metabolism. However, before they can be safely used, it is necessary to assess their mechanisms of action and potential side effects. This paper focuses on the metabolic potential of natural compounds to modulate telomerase and telomere biology and thus prevent senescence and skin aging. Keywords: telomerase; telomeres; natural compounds; senescence; skin aging; antioxidants Citation: Jacczak, B.; Rubi´s,B.; Toto´n,E. Potential of Naturally Derived Compounds in Telomerase and Telomere Modulation in Skin 1.