DOCSLIB.ORG

Mechanistic Studies of Inhibitors of Dna Replication

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanistic Studies of Inhibitors of Dna Replication MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION RESTART PATHWAYS IN NEISSERIA GONORRHOEAE Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Chemistry By Dasharatha Radha Krishna C. Aduri UNIVERSITY OF DAYTON Dayton, Ohio December, 2013 MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION RESTART PATHWAYS IN NEISSERIA GONORRHOEAE Name: Aduri, Dasharatha Radha Krishna C. APPROVED BY: ________________________________________________ Matthew E. Lopper, Ph.D. Faculty Advisor Assistant Professor Department of Chemistry ________________________________________________ Garry Crosson, Ph.D. Committee Member Assistant Professor Department of Chemistry ________________________________________________ Mark B. Masthay, Ph.D. Committee Member Associate Professor, Chair Department of Chemistry ________________________________________________ Shawn M. Swavey, Ph.D. Committee Member Associate Professor Department of Chemistry ii ABSTRACT MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION RESTART PATHWAYS IN NEISSERIA GONORRHOEAE Name: Aduri, Dasharatha Radha Krishna C. University of Dayton Advisor: Dr. Matthew E. Lopper, Ph.D. Complete and faithful replication of a cell’s genetic information is an essential process. Many enzymes are involved in the process of successfully duplicating a cell’s genetic information. Helicases, DNA polymerases, ligases, nucleases, and DNA binding proteins all play a role in DNA replication. However, the integrity of these enzymes can be compromised when they encounter DNA damage, which in general could be caused by chemical mutagens, ionizing radiations, or reactive oxidative species. Bacterial cells use a pathway called “DNA replication restart” to resume DNA replication following a disruptive encounter of the DNA replication enzymes with DNA damage. This pathway is catalyzed by primosome proteins, including PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The importance of DNA replication restart for bacterial cell survival is demonstrated by the inability of strains that carry mutations in key primosome genes to grow and resist DNA damaging agents. iii Furthermore, this pathway is specific for bacterial cells: human cells don’t use the same replication restart pathway and they don’t encode genes for the primosome proteins that function in bacteria. Since DNA replication restart pathways are essential for bacterial cell growth and survival and are notably absent in human cells, we seek to answer the following question: can bacterial DNA replication restart pathways be targeted with novel antibacterial compounds? In order to answer this question, we have developed an enzyme based assay for high-throughput inhibitor screening to identify compounds that block the function of the primosome proteins PriA and PriB. Several interesting lead compounds have already been identified from the preliminary screening. In this study, the lead compounds have been validated as legitimate inhibitors and characterized with respect to their potency and mechanism of action. iv Dedicated to my family and friends. v ACKNOWLEDGEMENTS First and foremost I would like to express my heartfelt gratitude to my advisor, Dr Matthew E. Lopper. I attribute the successful completion of my thesis project and writing of my thesis to his invaluable guidance, effort, and encouragement. Starting life as a research student one simply could not wish for a better or friendlier supervisor. His enthusiasm for biochemistry, his communication and leadership skills will always be an inspiration to me. I would like to thank my committee numbers Dr. Mark B. Masthay, Dr. Shawn M. Swavey and Dr. Garry Crosson for their support. I am grateful to my instructors Dr. David Johnson, Dr. Shawn Swavey, Dr. Gary Crosson, Dr. Mark Masthay and Dr. Matthew Lopper for their invaluable teaching and guidance. I specially thank Dr. Kevin Church for his help in selecting courses. In addition, I would also like to offer my sincerest gratitude to Paula Keil, Dr. Rochael Swavey for helping me with my teaching duties. I sincerely appreciate Connie Schell and Margaret Goodrich for being patient with me and sending reminders whenever I forgot to complete any office formality. On the whole I would like to thank Chemistry Department at University of Dayton for supporting me throughout my masters. vi TABLE OF CONTENTS ABSTRACT ……………………………………………………………………iii DEDICATION …………………………………………………………………v ACKNOWLEDGEMENTS ……………………………………………………vi LIST OF FIGURES …………………………………………………………….ix LIST OF TABLES ……………………………………………………………...xi CHAPTER I INTRODUCTION ………………………………………………..1 CHAPTER II METHODS ……………………………………………………....6 II-1 Purification of N. gonorrhoeae PriA ………………………………….....6 II-2 Purification of N. gonorrhoeae PriB ………………………………….....7 II-3 Purification of E. coli PriA ……………………………………………....8 II-4 Construction of Fork 2 DNA…………….……………………………....8 II-5 Helicase assays…………………………………………………………...9 II-6 ATP hydrolysis assays………………….………………………………..11 II-7 Crystallization ……………………………………………….…………..13 vii II-8 Determination of IC50 value ……………………………………………14 CHAPTER III RESULTS ……………………………………………………...16 III-1 Effect of Inhibitor F0298-0039 ………………………………………..16 III-2 Effect of Inhibitor F0359-0046 ………………………………………..18 III-3 Effect of Inhibitor F0683-0441 ………………………………………..20 III-4 Effect of Inhibitor F2018-1489 ………………………………………..22 III-5 Effect of Inhibitor F0683-0207 ………………………………………..24 III-6 Mechanism of the Inhibition of DNA replication restart process of Neisseria gonorrhoeae by F0683-0207…………………………………………….26 III-6-1 ATP hydrolysis experiments- DNA titrations ………….…………….27 III-6-2 ATP hydrolysis experiments- ATP titrations…………………………30 III-7 Effect of F0683-0207 on E. coli PriA…………………………………..32 III-8 Determining the activity of F0683-0207 on E. coli (MG 1655)………..33 III-9 Crystallization ………………………………………………………….35 CHAPTER IV CONCLUSIONS ………………………………………………40 REFERENCES …………………………………………………………………42 viii LIST OF FIGURES Figure 1: DNA replication process………………………………….……………2 Figure 2a: Unwinding of duplex DNA by PriA helicase ………………………..9 Figure 2b: Decrease in the fluorescence polarization value …………………….10 Figure 3: PriA catalyzed ATP regeneration cycle ……………………………….13 Figure 4: Structure of the Inhibitor F0298-0039 ………………………………...16 Figure 5a: Average data of the effect of F0298-0039 on N. gon PriA……..........17 Figure 5b: Average data of the effect of F0298-0039 on N. gon PriA and PriB...17 Figure 6: Structure of the Inhibitor F0359-0046………………………………….18 Figure 7a: Average data of the effect of F0359-0046 on N. gon PriA …………..19 Figure 7b: Average data of the effect of F0359-0046 on N. gon PriA and PriB…19 Figure 8: Structure of the Inhibitor F0683-0441………………………………….20 Figure 9: Average data of the effect of F0683-0441 on N. gonorrhoeae PriA ….21 Figure 10: Data showing the effect of F0683-0441 on intact DNA ……………..22 ix Figure 11: Structure of the Inhibitor F2018-1489 ………………………………..22 Figure 12: Data showing the effect of F2018-1489 on N. gon PriA……………...23 Figure 13: Data showing the effect of F2018-1489 intact DNA ………………...24 Figure 14: Structure of the Inhibitor F0683-0207 ………………………………...24 Figure 15a: Average data of the effect of F0683-0207 on N. gon PriA….………25 Figure 15b: Average data of the effect of F0683-0207 on N. gon PriA and PriB..25 Figure 16: DNA titrations…………………………………………………………29 Figure 17: ATP titrations ………………….………………………………………31 Figure 18: Plot displaying the effect of inhibitor F0683-0207 on E. coli PriA …...33 Figure 19: Plot displaying the growth rate of E. coli ……………………………...34 Figure 20: Tray setup for Hampton Crystal Screen ………………………………..36 Figure 21: Tray setup for Hampton Crystal Screen 2. …………………………….37 Figure 22: Tray setup for Hampton Index screen .……………………………..…..39 x LIST OF TABLES Table 1: Effect of mixed inhibitor on apparent Vmax and Km values……………..29 Table 2: DNA titrations: Kinetic parameters in the presence and absence of Inhibitor F0683-0207……………………………………………...……….………30 Table 3: ATP titrations: Kinetic parameters in the presence and absence of Inhibitor F0683-0207……………………………………….……………………...31 Table 4: Hampton Crystal Screen scoring sheet …………………………………..36 Table 5: Hampton Crystal Screen 2 scoring sheet……………………..…………..37 Table 6: Hampton Index Screen scoring sheet…………………………………….38 xi CHAPTER I INTRODUCTION The most essential aspect for the existence, survival and propagation of life is DNA replication and this involves the duplication of cellular genome. In bacteria, this process is continually disrupted by obstructions on the DNA template such as single stranded nicks, double stranded breaks, and oxidized bases which arise due to environmental or cellular factors1. The progress of the DNA replication machinery (replisome) is hindered by these barriers, causing it to stall or derail from the DNA template2. To ensure continuation of the DNA replication process, bacteria have evolved “DNA replication restart pathways” which reload and reactivate the DNA replisome onto a repaired DNA replication fork3. In E. coli, one of the several major pathways to activate stalled replication forks is DnaA-catalyzed origin-dependent initiation of DNA replication4 (Figure 1). This is a very carefully regulated, sequence-specific event which is catalyzed by the initiator protein, DnaA. DnaA recognizes and binds to a replication origin site and recruits the replicative helicase and the helicase loader protein, DnaB and DnaC, respectively. The replicative helicase unwinds the
Load more

Recommended publications
  • Initiation of Enzymatic Replication at the Origin of the Escherichia
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3954-3958, June 1985 Biochemistry Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: Primase as the sole priming enzyme (DNA/orC/plasmids) ARIE VAN DER ENDEt, TANIA A. BAKER, TOHRU OGAWA*, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Arthur Kornberg, January 28, 1985 ABSTRACT The enzymatic replication of plasmids con- MATERIALS AND METHODS taining the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme DNAs and Reagents. pCM959 (4) was a gift from M. Meijer priming systems: primase alone, RNA polymerase alone, or (University of Amsterdam, The Netherlands); pTOA7 (T. both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Ogawa) was constructed by inserting the Hae II-Acc I Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, oriC-containing fragment from M13oriC26 (7) via EcoRI 3562-3566). At certain levels of auxiliary proteins linkers into EcoRI-cleaved pMAPCdSG10, a deletion deriva- (topoisomerase I, protein HU, and RNase H), the solo primase tive of pBR327 (W. A. Segraves, personal communication); system is efficient and responsible for priming synthesis of all pSY317, M13oriC26, M13oriC2LB5, and M13AE101 are DNA strands. Replication of oriC plasmids is here separated described in Table 1 and elsewhere (3, 7). Tricine, creatine into four stages: (i) formation of an isolable, prepriming phosphate, ribo- and deoxyribonucleoside triphosphates complex requiring oriC, dnaA protein, dnaB protein, dnaC (rNTPs and dNTPs) were from Sigma; a-32P-labeled dTTP, protein, gyrase, single-strand binding protein, and ATP; (ii) rATP, rUTP, rGTP, and rCTP (>400 Ci/mmol; 1 Ci = 37 formation of a primed template by primase; (iii) rapid, GBq) were from Amersham.
    [Show full text]
  • Crystallographic Studies of the E. Coli DNA Replication Restart Primosome
    A Thesis entitled Crystallographic studies of the E. coli DNA replication restart primosome by Aude E. Izaac Submitted as partial fulfillment of the requirements for the Master of Science in Chemistry Adviser: Dr. Timothy C. Mueser Graduate School The University of Toledo May 2005 Copyright © 2005 This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Crystallographic studies of the E. coli DNA replication restart primosome Aude Izaac Submitted as partial fulfillment of the requirements for the Master of Science in Chemistry The University of Toledo May 2005 Understanding the mechanisms of DNA replication has been one of the main research topics in biochemistry for decades, due to the tremendous potential applications involved, such as medical treatments and better knowledge of the biological evolution of organisms. In this perspective, macromolecular X-ray crystallography provides a very powerful tool. By solving the 3-dimensional structure of the proteins involved in replication mechanisms, one can get a good insight at complicated biological systems at the atomic level. It is also important to study the protein-protein and protein-substrate interactions when they form biological complexes. iii This work focused on studying several proteins which are associated with the assembly of the replication restart primosome in Escherichia coli, during the repair of damaged DNA. The crystallization of PriA, the most important of the restart primosome proteins, was investigated. Two truncations of PriA (the PriA N and PriA NI domains) were expressed, purified and characterized to be prepared for crystallization.
    [Show full text]
  • Replication Fork Assembly at Recombination Intermediates Is Required for Bacterial Growth
    Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3552–3555, March 1999 Biochemistry Replication fork assembly at recombination intermediates is required for bacterial growth i JOING LIU†,LIEWEI XU‡,STEVEN J. SANDLER§, AND KENNETH J. MARIANS†‡¶ †Molecular Biology Graduate Program and ‡Biochemistry and Structural Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, NY 10021; §Department of Microbiology, University of Massachusetts at Amherst, Amherst, MA 01003; and ¶Molecular Biology Program, Memorial Sloan–Kettering Cancer Center, New York, NY 10021 Edited by Charles M. Radding, Yale University School of Medicine, New Haven, CT, and approved January 27, 1999 (received for review November 16, 1998) ABSTRACT PriA, a 3* 3 5* DNA helicase, directs assem- (12). Single-stranded DNA-binding protein (SSB) was purified bly of a primosome on some bacteriophage and plasmid DNAs. according to Minden and Marians (13). The DNA polymerase Primosomes are multienzyme replication machines that con- III holoenzyme was reconstituted either from Pol III* and b tribute both the DNA-unwinding and Okazaki fragment- subunit as described by Wu et al. (14) or from purified subunits priming functions at the replication fork. The role of PriA in as described by Marians et al. (15). chromosomal replication is unclear. The phenotypes of priA Preparation of D Loop Template DNA. A 100-nt-long oli- null mutations suggest that the protein participates in repli- gonucleotide having the sequence 59-ACATACATAAAGG- cation restart at recombination intermediates. We show here TGGCAACGCCATTCGAAATGAGCTCCATATGCTAG- that PriA promotes replication fork assembly at a D loop, an CTAGGGAGGCCCCCGTCACAATCAATAGAAAATT- intermediate formed during initiation of homologous recom- CATATGGTTTACCAGCGC-39 was annealed to f1R408 bination.
    [Show full text]
  • Crystal Structure of Prib, a Component of the Escherichia Coli Replication Restart Primosome
    Structure, Vol. 12, 1967–1975, November, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.09.004 Crystal Structure of PriB, a Component of the Escherichia coli Replication Restart Primosome Matthew Lopper,1 James M. Holton,2 in vitro conversion of bacteriophage φX174 single- and James L. Keck1,* stranded DNA (ssDNA) to its duplex replicative form 1Department of Biomolecular Chemistry (Schekman et al., 1975). Historically, this system pro- University of Wisconsin Medical School vided a model for lagging strand DNA synthesis wherein 550 Medical Sciences Center the role of the primosome is to repeatedly synthesize 1300 University Avenue short RNA oligonucleotides along the DNA template to Madison, Wisconsin 53706 prime Okazaki fragment synthesis. The seven proteins 2 Physical Biosciences Division comprising the primosome, PriA, PriB, PriC, DnaT, Lawrence Berkeley National Laboratory DnaB, DnaC, and DnaG, were isolated biochemically University of California, Berkeley based on this system (Schekman et al., 1975; Wickner Berkeley, California 94720 and Hurwitz, 1974). Subsequently, it was shown that E. coli possesses two primosomes: one that catalyzes replisome assembly at the bacterial origin of replication Summary (oriC) and involves three of the defined primosome pro- teins (DnaB, DnaC, and DnaG), and a second that initi- Maintenance of genome stability following DNA dam- ates replication at the φX174 origin (primosome assem- age requires origin-independent reinitiation of DNA bly site, pas) and involves all seven proteins. While DnaB replication at repaired replication forks. In E. coli, PriA, (the replicative helicase), DnaC (the helicase loading PriB, PriC, and DnaT play critical roles in recognizing protein), and DnaG (primase) were established as essen- repaired replication forks and reloading the replisome tial factors in chromosomal DNA replication initiating at onto the template to reinitiate DNA replication.
    [Show full text]
  • Ordered Association of Helicase Loader Proteins with the Bacillus Subtilis Origin of Replication in Vivo
    Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Smits, Wiep Klaas, Alexi I. Goranov, and Alan D. Grossman. “Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo.” Molecular Microbiology 75.2 (2010): 452–461. As Published http://dx.doi.org/10.1111/j.1365-2958.2009.06999.x Publisher Wiley Blackwell (Blackwell Publishing) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/73616 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike 3.0 Detailed Terms http://creativecommons.org/licenses/by-nc-sa/3.0/ NIH Public Access Author Manuscript Mol Microbiol. Author manuscript; available in PMC 2011 January 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Mol Microbiol. 2010 January ; 75(2): 452±461. doi:10.1111/j.1365-2958.2009.06999.x. Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo Wiep Klaas Smits, Alexi I. Goranov, and Alan D. Grossman* Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Summary The essential proteins DnaB, DnaD, and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order.
    [Show full text]
  • AFM Investigations of Critical Interactions in the Bacillus Primosome and Cryogenic AFM a New Tool for Structural Biology
    AFM investigations of critical interactions in the Bacillus primosome and Cryogenic AFM a new tool for structural biology Ian James Turner BSc (Hons), MSc Thesis submitted to The University of Nottingham for the degree of Doctor of Philosophy September 2005 This thesis is dedicated to my mum and dad; I know what you have been through to get me here. 2 Abstract In this thesis for the first AFM has been employed for the high resolution imaging of a protein assembly. The DnaB-DnaG Helicase-Primase interaction in Bacillus is the key reaction that causes the switch from primase mode to polymerisation mode. This assembly was imaged using the AFM to a sub- molecular resolution revealing structural detail of the interaction. It is shown that the binding of the primase causes the structure of the helicase to switch from a hexamer to a trimer of dimers with one primase molecule bound to each dimer; also the existance of sub-populations with one and two primases bound suggests a sequential mode of binding. Recently crystallography data has been published that confirms the structural observations generated by AFM here. This is the first time that AFM and crystallography data have been used concurrently to solve the molecular structure of a protein assembly and it shows the potential application of AFM for sub-molecular resolution imaging of other protein assemblies. The role of DnaD in the Bacillus primosome is well established, however, its exact function was unknown. In this thesis AFM was applied to help solve this biomolecular problem, it revealed that DnaD has a pivotal role in early primosome assembly, ‘opening up’ the DNA allowing other components of the cascade to bind.
    [Show full text]
  • Rnase HI Is Essential for Survival of Mycobacterium Smegmatis
    RESEARCH ARTICLE RNase HI Is Essential for Survival of Mycobacterium smegmatis Alina E. Minias1*, Anna M. Brzostek1, Malgorzata Korycka- Machala1, Bozena Dziadek2, Piotr Minias3, Malini Rajagopalan4, Murty Madiraju4, Jaroslaw Dziadek1* 1 Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland, 2 Department of Immunoparasitology, University of Lodz, Lodz, Poland, 3 Department of Teacher Training and Biodiversity Studies, University of Lodz, Lodz, Poland, 4 Department of Microbiology, University of Texas Health Center at Tyler, Tyler, Texas, United States of America * [email protected] (AM); [email protected] (JD) Abstract RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleo- tides are present. Here we investigated the importance of RNase H type I encoding genes OPEN ACCESS for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H Citation: Minias AE, Brzostek AM, Korycka- Machala M, Dziadek B, Minias P, Rajagopalan M, et al. (2015) type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis ge- RNase HI Is Essential for Survival of Mycobacterium nome without affecting the growth rate of the mutant. Further, we demonstrate that deletion smegmatis. PLoS ONE 10(5): e0126260. of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Final- doi:10.1371/journal.pone.0126260 ly, we question the possibility of existence of RNase HI related alternative mode of initiation Academic Editor: Anil Kumar Tyagi, University of of DNA replication in M.
    [Show full text]
  • Mechanism of Staphylococcal Multiresistance Plasmid Replication Origin Assembly by the Repa Protein
    Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein Maria A. Schumachera,1, Nam K. Tonthata, Stephen M. Kwongb, Naga babu Chinnama, Michael A. Liub, Ronald A. Skurrayb, and Neville Firthb aDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710; and bSchool of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia Edited by James M. Berger, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 15, 2014 (received for review April 1, 2014) The staphylococcal multiresistance plasmids are key contributors (16). In Gram-negative bacteria the primosome includes DnaG to the alarming rise in bacterial multidrug resistance. A conserved primase, the replicative helicase (DnaB), and DnaC (17). Rep- replication initiator, RepA, encoded on these plasmids is essential lication initiation in Gram-positive bacteria involves DnaG pri- for their propagation. RepA proteins consist of flexibly linked mase and helicase (DnaC) and the proteins DnaD, DnaI, and N-terminal (NTD) and C-terminal (CTD) domains. Despite their essen- DnaB (18–22). DnaD binds first to DnaA at the origin. This is tial role in replication, the molecular basis for RepA function is followed by binding of DnaI/DnaB and DnaG, which together unknown. Here we describe a complete structural and functional recruit the replicative helicase (23, 24). Instead of DnaA, plas- dissection of RepA proteins. Unexpectedly, both the RepA NTD and mids encode and use their own specific replication initiator CTD show similarity to the corresponding domains of the bacterial binding protein. Structures are only available for RepA proteins primosome protein, DnaD. Although the RepA and DnaD NTD both (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria.
    [Show full text]
  • The Pria Gene Encoding the Primosomal Replicative N' Protein of Escherichia Coil Eui HUM LEE*, HISAO Masaitt, GEORGE C
    Proc. Natl. Acad. Sci. USA Vol. 87, pp. 4620-4624, June 1990 Biochemistry The priA gene encoding the primosomal replicative n' protein of Escherichia coil Eui HUM LEE*, HISAO MASAItt, GEORGE C. ALLEN, JR.*, AND ARTHUR KORNBERG* *Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA 94305-5307; and tDNAX Research Institute of Molecular and Cellular Biology, 901 California Street, Palo Alto, CA 94304-1104 Contributed by Arthur Kornberg, April 2, 1990 ABSTRACT The Escherichia coli gene encoding protein n' and used according to the manufacturer's instructions. has been isolated and named priA for primosomal protein A. Highly purified DNA replication proteins were as described Protein n' is absolutely required for the conversion of single- (10). Plasmid pTZ18R was from Pharmacia LKB. The pGP1-2 stranded 4X174 DNA to the duplex replicative form in an in and pT7-6 plasmids were kindly provided by S. Tabor (Har- vitro-reconstituted system. The gene maps to 88.7 minutes on vard Medical School). the chromosome adjacent to the cytR locus. Soluble protein Reagents were: unlabeled deoxynucleotide triphosphates extracts from cells harboring the priA gene on a multicopy and ribonucleoside triphosphates (Pharmacia LKB); [y- plasmid contained 45-fold more n' replication activity than 32P]ATP, deoxyadenosine 5'-[a-T5S]thio]triphosphate, and wild-type extracts. Enhanced overproduction of >1000-fold [a-32P]dTTP (Amersham); bovine serum albumin (Pentax Fr was achieved by replacing the natural Shine-Dalgarno se- V; Sigma); prokaryotic DNA-directed translation kit (Amer- quence with that ofthe phage T7 410 gene and placing thispriA sham); Sequenase DNA sequencing kit (United States Bio- under the control of the T7 phage promoter and RNA poly- chemical); Erase-a-Base system kit (Promega).
    [Show full text]
  • Structural Basis for the Interaction of SARS-Cov-2 Virulence Factor Nsp1 with Pol - Primase
    bioRxiv preprint doi: https://doi.org/10.1101/2021.06.17.448816; this version posted June 19, 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 4.0 International license. Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol a - Primase Mairi L. Kilkenny1, Charlotte E. Veale1, Amir Guppy1, Steven W. Hardwick1, Dimitri Y. Chirgadze1, Neil J. Rzechorzek1,*, Joseph D. Maman1, Luca Pellegrini1 1 Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK *Current address: The Francis Crick Institute, London, NW1 1AT, UK bioRxiv preprint doi: https://doi.org/10.1101/2021.06.17.448816; this version posted June 19, 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 4.0 International license. Abstract The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus – the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic – are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase a - primase complex or primosome – responsible for initiating DNA synthesis in genomic duplication – was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection.
    [Show full text]
  • Replication (Prepriming/Protein N'/Dtaab Protein/Primase/Supercoiling) ROBERT L
    Proc. NatL Acad. SciL USA Vol. 78, No. 3, pp. 1436-1440, March 1981 Biochemistry Conservation of the primosome in successive -stages of 4X'174 DNA- replication (prepriming/protein n'/dtaaB protein/primase/supercoiling) ROBERT L. Low, KEN-ICHI ARAI*, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University'School of Medicine, Stanford, California 94305 Contributed by Arthur Kornberg, November 17, 1980 ABSTRACT Synthesis of a complementary strand to- match by primosome action of a ssDNA -- RF system identical to that the single-stranded, circular, viral (+) DNA.strand of phage used to initiate synthesis of the parental RF (16). 4+X174 creates a parental duplex circle (replicative form, RF). This synthesis is initiated by the assembly and action of a priming In this study, we have obtained physical and functional evi- system, called the primosome [Arai, K. &Kornberg, A (1981) Proc. dence that major components of the primosome (i.e., n' pro- NatL Acad. Sci USA'78, 69-73; Arai, K.,.Low, R. L. & Kornberg, tein, dnaB protein, and primase) isolated by gel filtration are A. (1981) Proc. NatL Acud. Sci. USA 78, 707-711]. Of the seven stably retained with the parental RF and function during RF proteins that participate in the assembly and function of the pri- duplication upon addition of proteins i, n", and dnaC. An even mosome, most all of the components remain even after the DNA more intact primosome isolated by sedimentation requires ad- duplex is completed and covalently sealed. Remarkably, the pri- mosome in the isolated RF obviates the need for superciling of dition ofonly protein i.
    [Show full text]
  • The Application of Thermophilic DNA Primase Ttdnag2 to DNA Amplifcation Received: 17 July 2017 D
    www.nature.com/scientificreports OPEN The application of thermophilic DNA primase TtDnaG2 to DNA amplifcation Received: 17 July 2017 D. Zhao1,2, Xiuqiang Chen1,2, Kuan Li3 & Yu V. Fu1,2 Accepted: 6 September 2017 For DNA replication in vivo, DNA primase uses a complementary single-stranded DNA template to Published: xx xx xxxx synthesize RNA primers ranging from 4 to 20 nucleotides in length, which are then elongated by DNA polymerase. Here, we report that, in the presence of double-stranded DNA, the thermophilic DNA primase TtDnaG2 synthesizes RNA primers of around 100 nucleotides with low initiation specifcity at 70 °C. Analysing the structure of TtDnaG2, we identifed that it adopts a compact conformation. The conserved sites in its zinc binding domain are sequestered away from its RNA polymerase domain, which might give rise to the low initiation specifcity and synthesis of long RNA segments by TtDnaG2. Based on these unique features of TtDnaG2, a DNA amplifcation method has been developed. We utilized TtDnaG2 to synthesize RNA primers at 70 °C after 95 °C denaturation, followed by isothermal amplifcation with the DNA polymerase Bst3.0 or phi29. Using this method, we successfully amplifed genomic DNA of a virus with 100% coverage and low copy number variation. Our data also demonstrate that this method can efciently amplify circular DNA from a mixture of circular DNA and linear DNA, thus providing a tool to amplify low-copy-number circular DNA such as plasmids. DNA replication is a complicated process in vivo1–3. It requires numerous components working together to syn- thesize new DNA strands.
    [Show full text]