1 Mechanistic Studies of DNA Replication, Lesion Bypass, And
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The Genetic Material: DNA the Central Dogma of Genetics
The Genetic material: DNA The Central Dogma of Genetics DNA transcription Reverse tititranscriptio RNA n translation Protein • A, T, G, C in DNA • A, U, G, C in RNA • DNA is double stranded • DNA has po lar ity 5’ to 3’ • A T base pair, G C base pair • RNA is single stranded, also has polarity , generally referred as upstream and downstream. In RNA: A U base pair G C base pair. This type of base pa iri ng i n RNA causes secon dary st ruct ure. 1. 5’ vs 3’ 2. Purines vs. Pyrimidines 3. A vs. G 4. C vs. T 5. Transitions vs. transversions AT/GC ratios and their applications • Genome composition and chtitiharacterization • Implications in sequencing • Primer design • PCR yields • … The Polarity of DNA Higher order organization of genomes Most chromosomal DNA does not code for proteins or RNAs: e.g., Human genome 3 billion base pairs 25,000 genes x 2,000 bp per gene =5x10= 5 x 107 bp 5 x 107/3 x 109 = 1.67% MlMolecu lar P rob es: The tools of molecular genetics Concept of probes • “For diagnostic tests, the agent that is used to detect the presence of a molecule in the sample”. • “A DNA sequence that is used to detect the presence o f a comp lement ary sequence by hybridization with a nucleic acid sample”. Need for probes • Screen for the gene of interest • Southern blot to understand genomic structure and gene copy numbers • Northern blot for analysis of RNA expression • Verification of allelic amplification in PCR • .. -
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 -
Distinct Co-Evolution Patterns of Genes Associated to DNA Polymerase III Dnae and Polc Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3*
Engelen et al. BMC Genomics 2012, 13:69 http://www.biomedcentral.com/1471-2164/13/69 RESEARCHARTICLE Open Access Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3* Abstract Background: Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results: Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co- evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion: DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world. -
Glycolytic Pyruvate Kinase Moonlighting Activities in DNA Replication
Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere To cite this version: Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere. Gly- colytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation. 2020. hal-02992157 HAL Id: hal-02992157 https://hal.archives-ouvertes.fr/hal-02992157 Preprint submitted on 10 Dec 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. Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans1, Matthaios Pitoulias2, Alexandria Holland2, Panos Soultanas2¶ and Laurent Janniere1¶ 1 : Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France 2 : Biodiscovery Institute, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK Short title: PykA moonlighting activity in DNA replication Key Words: DNA replication; replication control; central carbon metabolism; glycolytic enzymes; replication enzymes; cell cycle; allosteric regulation. ¶ : Corresponding authors Laurent Janniere: [email protected] Panos Soultanas : [email protected] 1 SUMMARY Cells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. -
The Role of Metabolites in the Link Between DNA Replication and Central Carbon Metabolism in Escherichia Coli
G C A T T A C G G C A T genes Article The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli Klaudyna Krause 1, Monika Maci ˛ag-Dorszy´nska 2 , Anna Wosinski 3, Lidia Gaffke 3 , Joanna Morcinek-Orłowska 1,3, Estera Rintz 3, Patrycja Biela´nska 3, Agnieszka Szalewska-Pałasz 1, Georgi Muskhelishvili 4 and Grzegorz W˛egrzyn 3,* 1 Department of Bacterial Molecular Genetics, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (K.K.); [email protected] (J.M.-O.); [email protected] (A.S.-P.) 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 80-822 Gdansk, Poland; [email protected] 3 Department of Molecular Biology, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (A.W.); lidia.gaff[email protected] (L.G.); [email protected] (E.R.); [email protected] (P.B.) 4 School of Natural Sciences, Agricultural University of Georgia, 0131 Tbilisi, Georgia; [email protected] * Correspondence: [email protected]; Tel.: +48-58-5236024 Received: 31 March 2020; Accepted: 16 April 2020; Published: 19 April 2020 Abstract: A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. -
Coordination Between Nucleotide Excision Repair And
RESEARCH ARTICLE Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria Asha Mary Joseph1, Saheli Daw1, Ismath Sadhir1,2, Anjana Badrinarayanan1* 1National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bangalore, India; 2Max Planck Institute for Terrestrial Microbiology, LOEWE Centre for Synthetic Microbiology (SYNMIKRO), Marburg, Germany Abstract Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication- independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner. *For correspondence: [email protected] Introduction Competing interests: The DNA damage is a threat to genome integrity and can lead to perturbations to processes of replica- authors declare that no tion and transcription. -
Coordination Between Nucleotide Excision Repair and Specialized Polymerase Dnae2 Action 2 Enables DNA Damage Survival in Non-Replicating Bacteria
bioRxiv preprint doi: https://doi.org/10.1101/2021.02.15.431208; this version posted February 15, 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. 1 Coordination between nucleotide excision repair and specialized polymerase DnaE2 action 2 enables DNA damage survival in non-replicating bacteria 3 4 5 6 7 Asha Mary Joseph, Saheli Daw, Ismath Sadhir and Anjana Badrinarayanan* 8 9 National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bellary Road, 10 Bangalore 560065, Karnataka, India, Phone: 91 80 23666547 11 *Correspondence to: [email protected] 12 13 Keywords 14 Caulobacter crescentus, DnaE2, DNA repair, error-prone polymerases, non-replicating cells, 15 nucleotide excision repair, single-cell imaging, fluorescence microscopy 16 17 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.15.431208; this version posted February 15, 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. 18 Abstract 19 Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which 20 employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models 21 suggest that activity of these polymerases is predominantly associated with ongoing replication, 22 functioning either at or behind the replication fork. -
A Multi-Disciplinary Investigation of Essential DNA Replication Proteins
A Multi-Disciplinary Investigation of Essential DNA Replication Proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Varun V. Gadkari Graduate Program in Biochemistry The Ohio State University 2017 Dissertation Committee: Dr. Zucai Suo, Advisor Dr. Jane Jackman Dr. Comert Kural Dr. Richard Swenson Copyrighted by Varun V. Gadkari 2017 Abstract An organism’s DNA is constantly under attack from various exogenous and endogenous DNA damaging agents. Thus, to assure survival, all living cells have evolved to maintain the genetic integrity of their DNA by various pathways. If left unrepaired, DNA damage sites, or “lesions” can block DNA replication by stalling DNA polymerases, the enzymes responsible for DNA replication. Ultimately, if a stalled replication fork is not rescued, the cell will undergo apoptosis. To bypass DNA lesions, organisms in all domains of life initiate a process known as Translesion DNA Synthesis (TLS). During TLS, the stalled replicative DNA polymerase is displaced by specialized Y-family DNA polymerases that are capable of efficiently bypassing various forms of DNA damage. While Y-family DNA polymerases are proficient in TLS, the fidelity of the process is a notable cause for concern. TLS mechanisms of the different Y-family DNA polymerases vary greatly, and often introduce mutations in the DNA which can lead to carcinogenesis. Thus, the activity of Y-family DNA polymerases must be strictly regulated. To this end all living organisms depend on evolutionarily conserved sliding DNA clamps which bind the DNA in a toroidal fashion, and slide along DNA during replication, serving as a scaffold for the DNA replication and repair machinery. -
A Model for DNA Polymerase Switching Involving a Single Cleft and the Rim of the Sliding Clamp
A model for DNA polymerase switching involving a single cleft and the rim of the sliding clamp Justin M. H. Heltzel1, Robert W. Maul1,2, Sarah K. Scouten Ponticelli, and Mark D. Sutton3 Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214 Edited by Sue Hengren Wickner, National Institutes of Health, Bethesda, MD, and approved June 2, 2009 (received for review March 30, 2009) The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagen- proteins involved in DNA replication, DNA repair, and cell cycle esis are managed by its interaction with the  sliding clamp. In the progression (9). A similar situation is true for many eukaryotic Pols structure reported by Bunting et al. [EMBO J (2003) 22:5883–5892], the (10). Taken together, these findings suggest that sliding clamps act C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while like traffic cops to coordinate the actions of different partners on residues V303–P305 reach over the dimer interface to contact the rim DNA. of the adjacent clamp protomer. Using mutant forms of these proteins One contact site between these partners and the clamp involves impaired for either the rim or the cleft contacts, we determined that a clamp-binding motif (CBM) present in the partners and a the rim contact was dispensable for Pol IV replication in vitro, while hydrophobic cleft located near the C-terminal tail of each clamp the cleft contact was absolutely required. Using an in vitro assay to protomer (11, 12). -
2-Sliding Clamp Accumulation in Live Escherichia Coli Cells
ARTICLE Received 8 Jul 2014 | Accepted 11 Nov 2014 | Published 18 Dec 2014 DOI: 10.1038/ncomms6820 OPEN Slow unloading leads to DNA-bound b2-sliding clamp accumulation in live Escherichia coli cells M. Charl Moolman1, Sriram Tiruvadi Krishnan1, Jacob W.J. Kerssemakers1, Aafke van den Berg1, Pawel Tulinski1, Martin Depken1, Rodrigo Reyes-Lamothe2, David J. Sherratt3 & Nynke H. Dekker1 The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli b2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both b2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single- molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of b2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous b2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism. 1 Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. 2 Department of Biology, McGill University, Montreal, Quebec, Canada H3G 0B1. 3 Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. Correspondence and requests for materials should be addressed to N.H.D. (email: [email protected]). NATURE COMMUNICATIONS | 5:5820 | DOI: 10.1038/ncomms6820 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. -
Phenotypic Characterization of Self-Assembling
PHENOTYPIC CHARACTERIZATION OF SELF-ASSEMBLING PROTEIN FRAGMENTS USING NEGATIVE DOMINANCE A Dissertation by ADRIENNE ELIZABETH ZWEIFEL Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2010 Major Subject: Biochemistry PHENOTYPIC CHARACTERIZATION OF SELF-ASSEMBLING PROTEIN FRAGMENTS USING NEGATIVE DOMINANCE A Dissertation by ADRIENNE ELIZABETH ZWEIFEL Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, James C. Hu Committee Members, Michael Polymenis Donald W. Pettigrew Michael Benedik Head of Department, Gregory D. Reinhart May 2010 Major Subject: Biochemistry iii ABSTRACT Phenotypic Characterization of Self-Assembling Protein Fragments Using Negative Dominance. (May 2010) Adrienne Elizabeth Zweifel, B.S., University of Missouri-Columbia Chair of Advisory Committee: Dr. James C. Hu Protein oligomerization provides a way for cells to modulate function in vivo. In this study, self-assembling protein fragments from ParC, DnaX, and proteins of unknown function were used to generate phenotypes in a dominant negative manner. These fragments were expressed as Thioredoxin (TRX) fusions under the control of the inducible araBAD promoter. Fragments chosen contain only the oligomerization domain of the protein, lacking the regions necessary for catalytic function. Fragments of ParC, a subunit of Topoisomerase (Topo) IV, generated fragment-specific phenotypes. Regions that expressed both the oligomerization domain and CTD of ParC (ParC206-752 and ParC332-752) yielded filamentous cells with several different nucleoid segregation phenotypes. Another ParC fragment containing only the oligomerization domain of ParC (ranging from 333-485) yields a recA-dependent septation defect in a subset of the population. -
A DNA Fragment Containing the Origin of Replication Of
Proc. Natl. Acad. Sci. USA Vol. 74, No. 7, pp. 2720-2724, July 1977 Biochemistry A DNA fragment containing the origin of replication of the Escherichia coli chromosome (bidirectional replication/dnaA and dnaC initiation mutants/restriction mapping) ROBERT C. MARSH* AND ABRAHAM WORCELt * Biology Program, The University of Texas at Dallas, P.O. Box 688, Richardson, Texas 75080; and t Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540 Communicated by Jerard Hurwitz, April 15, 1977 ABSTRACJ A 38 kilobase pair region of the Escherichia coli mg of Na2SO4, and 12.1 g of Tris. The medium additionally K12 chromosome containing the replication origin has been contained 0.2% glucose, 0.4% casamino acids, and 40 ,gg of physically mapped with restriction endonucleases EcoRI and thymine per ml. HindIII. Replication starts within or very near a 1.3 kilobase pair HindIII fragment in the middle of this region and proceeds Radioactive Labeling of Origin Region and Isolation of outward in both directions with apparently equal speed. This DNA. To obtain synchronized initiation of replication, we grew pattern was observed in both dnaA and dnaC temperature- cultures of PC2 and PC5 at 280C to 0.25 OD600 and then shifted sensitive (ts) initiation mutants at the start of the synchronous them to 40°. After 1 hr. the cultures were rapidly cooled to 280 round of replication which occurs after downshift from the and 10-ml aliquots taken at 1- or 2-min intervals for pulse la- nonpermissive to the permissive temperature. beling with 0.4 ml of [3H]thymidine (0.5 mCi/ml and 60 Ci/ The origin of replication of the Escherichia coli chromosome mmol, Schwarz/Mann).