DOCSLIB.ORG

Bacteriophage PRD1 DNA Polymerase: Evolution of DNA Polymerases (Lipid-Containing Phage/Protein-Primed DNA Replication) GUHUNG JUNG, MARK C

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bacteriophage PRD1 DNA Polymerase: Evolution of DNA Polymerases (Lipid-Containing Phage/Protein-Primed DNA Replication) GUHUNG JUNG, MARK C Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8287-8291, December 1987 Biochemistry Bacteriophage PRD1 DNA polymerase: Evolution of DNA polymerases (lipid-containing phage/protein-primed DNA replication) GUHUNG JUNG, MARK C. LEAVITT, JUI-CHENG HSIEH, AND JUNETSU ITO Department of Microbiology and Immunology, The University of Arizona Health Sciences Center, Tucson, AZ 85724 Communicated by Carl S. Marvel, August 10, 1987 ABSTRACT A small lipid-containing bacteriophage PRD1 sensitive to the drug aphidicolin, which is a specific inhibitor specifies its own DNA polymerase that utilizes terminal protein of eukaryotic DNA polymerases a and A (10, 16). as a primer for DNA synthesis. The PRD1 DNA polymerase In this communication, we report the nucleotide sequence gene has been sequenced, and its amino acid sequence has been and the deduced amino acid sequence of PRD1 DNA deduced. This protein-primed DNA polymerase consists of 553 polymerase.* We have compared the amino acid sequence of amino acid residues with a calculated molecular weight of this polymerase with those of other DNA polymerases and 63,300. Thus, it appears to be the smallest DNA polymerase found that PRD1 DNA polymerase, the smallest known DNA ever isolated from prokaryotic cells. Comparison of the PRD1 polymerase possessing a proofreading function, has partial DNA polymerase sequence with other DNA polymerase se- homology with many other DNA polymerases including quences that have been published yielded segmental but sig- phage T4 DNA polymerase. These results together with those hificant homologies. These results strongly suggest that many of others (17-22) suggest strongly that many prokaryotic and pi-okaryotic and eukaryotic DNA polymerase genes, regardless eukaryotic DNA polymerase genes, regardless of size, have of size, have evolved from a common ancestral gene. The evolved from a common ancestral gene. The results also results further indicate that those DNA polymerases that use suggest that DNA polymerases that use either an RNA or either an RNA or protein primer are related. We propose to protein primer have arisen from a common ancestral progen- classify DNA polymerases on the basis of their evolutionary itor. relatedness. MATERIALS AND METHODS Since DNA contains the genetic information of an organism, accurate DNA replication is one ofthe most important events DNA Sequencing. A recombinant plasmid (clone 3) that ofthe life cycle ofan organism. DNA polymerases are the key contains PRD1 genes 1 and 8 was kindly provided by L. enzymes catalyzing the accurate replication of DNA. How- Mindich (Public Health Research Institute ofthe City ofNew ever, none of the known DNA polymerases can initiate de York) (13). The plasmid DNA was first cleaved with Pst I, novo synthesis of a DNA chain without a primer (1). Gen- and the resulting DNA fragment containing genes I and 8 of erally, small RNA molecules are used as primers. These PRD1 was recloned into the bacteriophage vector M13mp9 provide a 3'-hydroxyl group for DNA chain elongation (1). (23). We then generated a nested set ofdeletions according to However, a different type of DNA polymerase has been the method of Dale et al. (24). Each set of deletions was identified in various systenis (2-7). These enzymes utilize sequenced by the dideoxy chain-termination method of proteins as primers for the initiation of DNA synthesis. Sanger et al. (25). For confirmation, some regions were Whereas these DNA polymerases are distinctive in the way sequenced by the method of Maxam and Gilbert (26). they initiate DNA synthesis, their mechanism for achieving Computation. Protein data base searchest were performed high fidelity of DNA replication appears to be similar to that by a FASTP microcomputer program (27). DNA polymerase of the RNA-primed DNA polymerases. Thus, all known nucleotide sequences were obtained from the GenBank data DNA polymerases acting at protein, primers also possess a baset and translated and screened against consensus se- 3'-+5' exonuclease activity, generally known as the editing quences with the use of the IBI (International Biotechnolo- function (8-10). gies, Inc.) sequence analysis system program. We have been studying bacteriophage PRD1 as a model RESULTS system for the initiation of linear DNA replication. PRD1 is a member of a group of lipid-containing phages that infect a Nucleotide Sequence and Predicted Amino Acid Sequence of wide variety ofGram-negative bacteria harboring PRD1 DNA Polymerase. In agreement with genetic studies by plasmids of McGraw et al. (13), nucleotide sequence analysis revealed the P, N, or W incompatibility type (11, 12). The genome of that there are only two major open reading frames at the left PRD1 is 14.7 kilobases long with terminal proteins covalently end of the PRD1 genome (Fig. 1). One open reading frame linked at both 5' ends (13). The linkage ofthe terminal protein corresponds to gene 8, the terminal protein gene consisting of to the PRD1 DNA involves a phosphodiester bond between 260 codons. The other one is gene 1, the DNA polymerase a tyrosine residue of the terminal protein and the 5'-terminal gene that consists of 554 codons (Fig. 1). The nucleotide se- dGMP (6). The PRD1 genome encodes a DNA polymerase quence of the PRD1 DNA polymerase gene along with the that catalyzes the formation of the terminal protein-dGMP complex as well as DNA chain elongation, analogous to the *The sequence reported in this paper is being deposited in the systems ofadenovirus (14, 15) and 429 (4,5). The PRD1 DNA EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- polymerase has been isolated and shown to possess a tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) 3'--5'exonuclease activity (10). It has also been shown to be (accession no. J03018). tProtein Identification Resource (1986) Protein Sequence Database (Natl. Biomed. Res. Found., Washington, DC), Release 9.0. T,he publication costs of this article were defrayed in part by page charge tNational Institutes of Health (1987) Genetic Sequence Databank: payment. This article must therefore be hereby marked "advertisement" GenBank (Research Systems Div., Bolt, Beranek, and Newman, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cambridge, MA), Tape Release 48.0. 8287 Downloaded by guest on September 24, 2021 8288 Biochemistry: Jung et al. Proc. Natl. Acad. Sci. USA 84 (1987) PRD1 GENOME residues and a high percentage (41.2%) of hydrophobic residues. The initiation codon ATG for the PRD1 DNA poly- merase gene is preceded by a region complementary to the 3' terminus of Escherichia coli 16S rRNA (29). Comparison of Amino Acid Sequences Between Phage PRD1 and Phage 429 DNA Polymerases. Our studies show that PRD1 and 429 DNA polymerases are the smallest known enzymes that possess 3'-5' exonuclease activity. Moreover, these two DNA polymerases utilize terminal proteins as primers for the initiation of DNA synthesis (4, 6, 9, 10). Because 429 infects Gram-positive bacteria and PRD1 grows on Gram-negative bacteria, the amino acid sequences of these two DNA polymerases were compared to discern the possible evolutionary relationship between these proteins. As Although the overall sequences are not strikingly homolo- gous, three regions are highly conserved between these two er DNA polymerases (Fig. 3). To determine whether these localized homologies are specific for protein-primed DNA FIG. 1. Genetic and physical map of the region of PRD1 that polymerases, we compared these amino acid sequences with contains the genes for DNA polymerase (gene 1) and terminal protein that of phage SP02 DNA polymerase that has a similar (gene 8). The genetic map was adapted from that of McGraw et al. molecular weight. The Bacillus temperate phage SP02 spec- (13). Arrows indicate the direction of translation. The open circles at ifies its own DNA polymerase (31), and the gene encoding both ends of the genome indicate terminal protein. this polymerase has been cloned and sequenced by Raden and Rutberg (31). The SP02 DNA polymerase has a molec- predicted amino acid sequence and the flanking sequences ular weight of 72,000 and does not initiate DNA synthesis by are shown in Fig. 2. The calculated molecular weight ofPRD1 a protein-priming mechanism (31). No substantial amino acid DNA polymerase is 63,300, which is in rough agreement with homology was found when the sequence for SP02 DNA the reported size (28). This protein contains an approximately polymerase was compared with the sequence for either 429 equal nurpber of acidic (14.2%) and basic (15.7%) amino acid or PRD1 DNA polymerases (data not shown). AAA CGC GGC TAT GGC AGC MG GGG GTT TAA GAT ATS| CCG CGC CGT TCC CGT AAA AAG GTG GAA TAT AA AT GCC LJ P R R S R K K V E Y K I A GCC '[T GAc 'Tr GM. ACT GAC CCT TTC AAG CAT GAC CGA ATC CCT AAA CCG TTT TCA TOG GGT 'FF TAT AAT GGC 15 A F D F E T D P F K H D R I P K P F S W G F Y N G GAA AT TAT MAA GAC TAT 'OG GOC GAT (;A TGC AlA GM CAG AvlT'FF1' '[AC'TG IT GAT ACC A'l'A GAA GAA CCG 40 E I Y K D) Y W G D D C I E Q F I Y W L D T I E E P CAC GOrr ATA [AC GmT CAT AAC GC GGC MG FTr GAT 'FITr CT[ r[T CrC ATG AMA TAC 1'IT CGC GGG AAA TG AAA 65 H V I Y A H N G G K F DI F L F L M K Y F R G K L K ATA G MT GGG CGT AAT IGT GAM GTA GAM CAC GGC ATO CAT AAA Ft CGC GAT AGT TAT GCA ATC CTG CCG GTG 90 I V N G K I L E V E H G I H K F R D S Y A I L P V CCG CrI' GC' GCC Gc, (;GAT GM MAG ATA GMAA NrIr GAT TAT ('C AAG ATG GMA AGG GM ACA CGC GAA CAG CAC AAG 115 P L A A S D E K I E I D Y G K M k R E 'r R k Q H K GCG GAA ANT 'WA GMAA 'Ac (G.A GG (GAT ''T G'rA ACC cTG CAT AM TG GTr TC 'rFA '-FI AMT1 GCT GAA T- 140 A E I L E Y L K G D C V 1' L H K M V S L F I A E F GGA MGlY.
Load more

Recommended publications
  • A Mutation in DNA Polymerase Α Rescues WEE1KO Sensitivity to HU
    International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int.
    [Show full text]
  • Human Glucokinase Gene
    Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7698-7702, August 1992 Genetics Human glucokinase gene: Isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus (glucose/metabolism/phosphorylation/structure4unctlon/chromosome 7) M. STOFFEL*, PH. FROGUELt, J. TAKEDA*, H. ZOUALItt, N. VIONNET*, S. NISHI*§, I. T. WEBER¶, R. W. HARRISON¶, S. J. PILKISII, S. LESAGEtt, M. VAXILLAIREtt, G. VELHOtt, F. SUNtt, F. lIRSt, PH. PASSAt, D. COHENt, AND G. I. BELL*"** *Howard Hughes Medical Institute, and Departments of Biochemistry and Molecular Biology, and of Medicine, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637; §Second Division of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-32, Japan; IDepartment of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; IlDepartment of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794; tCentre d'Etude du Polymorphisme Humain, 27 rue Juliette Dodu, and Service d'Endocrinologie, H6pital Saint-Louis, 75010 Paris, France; and tG6ndthon, 1 rue de l'Internationale, 91000 Evry, France Communicated by Jean Dausset, May 28, 1992 ABSTRACT DNA polymorphisms in the glucokinase gene by maintaining a gradient for glucose transport into these cells have recently been shown to be tightly linked to early-onset thereby regulating hepatic glucose disposal. In (3 cells, glu- non-insulin-dependent diabetes mellitus in "80% of French cokinase is believed to be part of the glucose-sensing mech- families with this form of diabetes. We previously identified a anism and to be involved in the regulation ofinsulin secretion.
    [Show full text]
  • Arthur Kornberg Discovered (The First) DNA Polymerase Four
    Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E.
    [Show full text]
  • Taming the Wild Rubisco: Explorations in Functional Metagenomics
    Taming the Wild RubisCO: Explorations in Functional Metagenomics DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian Hurin Witte, M.S. Graduate Program in Microbiology The Ohio State University 2012 Dissertation Committee : F. Robert Tabita, Advisor Joseph Krzycki Birgit E. Alber Paul Fuerst Copyright by Brian Hurin Witte 2012 Abstract Ribulose bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39) (RubisCO) is the most abundant protein on Earth and the mechanism by which the vast majority of carbon enters the planet’s biosphere. Despite decades of study, many significant questions about this enzyme remain unanswered. As anthropogenic CO2 levels continue to rise, understanding this key component of the carbon cycle is crucial to forecasting feedback circuits, as well as to engineering food and fuel crops to produce more biomass with few inputs of increasingly scarce resources. This study demonstrates three means of investigating the natural diversity of RubisCO. Chapter 1 builds on existing DNA sequence-based techniques of gene discovery and shows that RubisCO from uncultured organisms can be used to complement growth in a RubisCO-deletion strain of autotrophic bacteria. In a few short steps, the time-consuming work of bringing an autotrophic organism in to pure culture can be circumvented. Chapter 2 details a means of entirely bypassing the bias inherent in sequence-based gene discovery by using selection of RubisCO genes from a metagenomic library. Chapter 3 provides a more in-depth study of the RubisCO from the methanogenic archaeon Methanococcoides burtonii.
    [Show full text]
  • Multiple Elements in the Upstream Glucokinase Promoter Contribute to Transcription in Insulinoma Cells KATHY D
    MOLECULAR AND CELLULAR BIOLOGY, Oct. 1992, p. 4578-4589 Vol. 12, No. 10 0270-7306/92/104578-12$02.00/O Copyright X 1992, American Society for Microbiology Multiple Elements in the Upstream Glucokinase Promoter Contribute to Transcription in Insulinoma Cells KATHY D. SHELTON, ALAN J. FRANKLIN, ANDRAS KHOOR,t JOSEPH BEECHEM, AND MARK A. MAGNUSON* Departments ofMolecular Physiology and Biophysics and ofMedicine, Vanderbilt University Medical School, Nashville, Tennessee 37232 Received 28 February 1992/Returned for modification 31 March 1992/Accepted 23 July 1992 1-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a ,8-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIlT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -280 and -1 bp con- tributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet 13 cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G).
    [Show full text]
  • DNA Polymerase I- Dependent Replication (Temperature-Sensitive Dna Mutants/Extragenic Suppression) OSAMI NIWA*, SHARON K
    Proc. Nati Acad. Sci. USA Vol. 78, No. 11, pp. 7024-7027, November 1981 Genetics Alternate pathways of DNA replication: DNA polymerase I- dependent replication (temperature-sensitive dna mutants/extragenic suppression) OSAMI NIWA*, SHARON K. BRYAN, AND ROBB E. MOSES Department ofCell Biology, Baylor College of Medicine, Houston, Texas 77030 Communicated by D. Nathans, July 10, 1981 ABSTRACT We have previously shown that someEscherichia proceed in the presence of a functional DNA polymerase I ac- coli [derivatives of strain HS432 (polAl, polB100, polC1026)] can tivity, despite a ts DNA polymerase III (6). replicate DNA at a restrictive temperature in the presence of a We report here that DNA replication in the parent strain polCts mutation and that such revertants contain apparent DNA becomes temperature-resistant with introduction ofDNA poly- polymerase I activity. We demonstrate here that this strain ofE. merase I activity but is ts in the absence of DNA polymerase colibecomes temperature-resistant upon the introduction ofa nor- I or presence of a ts DNA polymerase I activity. We conclude mal gene for DNA polymerase I or suppression of the polAl non- that this strain contains a sense mutation. Such temperature-resistant phenocopies become mutation (pcbA-) that allows repli- temperature-sensitive upon introduction of a temperature-sensi- cation to be dependent on DNA polymerase I polymerizing tive DNA polymerase I gene. Our results confirm that DNA rep- activity. This locus can be transduced to other E. coli strains and lication is DNA polymerase I-dependent in the temperature-re- again exerts phenotypic suppression of the polCts mutation in sistant revertants, indicating that an alternative pathway of the presence of DNA polymerase I.
    [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]
  • The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN]
    The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN] Jixiang Zhang, Shaojun Xie, Jinkui Cheng, Jinsheng Lai, Jian-Kang Zhu, and Zhizhong Gong* State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China (J.Z., J.C., Z.G.); Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (S.X., J.-K.Z.); Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47906 (S.X., J.-K.Z.); and State Key Laboratory of Agrobiotechnology, China National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China (J.L.) ORCID IDs: 0000-0002-1641-8650 (J.Z.); 0000-0002-6719-9814 (S.X.); 0000-0001-5134-731X (J.-K.Z.). DNA polymerase d plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase d, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase a. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels.
    [Show full text]
  • A Coupled Complex of T4 DNA Replication Helicase (Gp41)
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14456–14461, December 1996 Biochemistry A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis (helicase–polymeraseyDNA unwindingyATPaseykineticsymacromolecular crowding) FENG DONG,STEVEN E. WEITZEL, AND PETER H. VON HIPPEL* Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1129 Contributed by Peter H. von Hippel, September 30, 1996 ABSTRACT We have developed a coupled helicase– determine how fast the helicase moves through the dsDNA in polymerase DNA unwinding assay and have used it to monitor carrying out the unwinding reaction. Furthermore the ‘‘prod- the rate of double-stranded DNA unwinding catalyzed by the uct’’ of such assays is, of course, simply two separated DNA phage T4 DNA replication helicase (gp41). This procedure can strands that will spontaneously rehybridize unless some means be used to follow helicase activity in subpopulations in systems is found to ‘‘trap’’ the products without interfering with the in which the unwinding-synthesis reaction is not synchronized reaction. Due to difficulties in achieving efficient and synchro- on all the substrate-template molecules. We show that T4 nized loading of the T4 DNA helicase (which has a fairly low replication helicase (gp41) and polymerase (gp43) can be affinity for DNA; see ref. 6), recently developed methods that assembled onto a loading site located near the end of a long are based on observing overall populations (7–9) have not double-stranded DNA template in the presence of a macro- been useful in studying the unwinding of dsDNA by the gp41 molecular crowding agent, and that this coupled ‘‘two- helicase.
    [Show full text]
  • Characterisation of the ATP-Dependent Phosphofructokinase Gene Family from Arabidopsis Thaliana
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 2401–2410 Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana Angelika Mustropha,1, Uwe Sonnewaldb, Sophia Biemeltb,* a Humboldt-University Berlin, Institute of Biology, AG Plant Physiology, Philippstrasse 13, 10115 Berlin, Germany b Friedrich-Alexander University Erlangen-Nuremberg, Department of Biochemistry, Staudtstrasse 5, 91058 Erlangen, Germany Received 1 March 2007; revised 15 April 2007; accepted 16 April 2007 Available online 30 April 2007 Edited by Mark Stitt addition to the widely distributed ATP-dependent phospho- Abstract Plants possess two different types of phosphofructo- kinases, an ATP-dependent (PFK) and a pyrophosphate-depen- fructokinase (PFK, EC 2.7.1.11), in plants and a number of dent form (PFP). While plant PFPs have been investigated in prokaryotes a second type of phosphofructokinase is present detail, cDNA clones coding for PFK have not been identified using pyrophosphate (PPi) instead of ATP as phosphoryl in Arabidopsis thaliana. Searching the A. thaliana genome re- donor (pyrophosphate-fructose-6-phosphate-phosphotransfer- vealed 11 putative members of a phosphofructokinase gene fam- ase, PFP, EC 2.7.1.90). Phosphorylation of Fru6P catalyzed by ily. Among those, four sequences showed high homology to the PFK is virtually irreversible in vivo, while PFP reacts near alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted equilibrium and catalyzes the reaction in both directions. In in elevated PFK, but not PFP activity after transient expression addition a third, ADP-dependent phosphofructokinase has in tobacco leaves suggesting that they encode Arabidopsis PFKs.
    [Show full text]
  • A Review of Isozymes in Cancer1
    Cancer Research VOLUME31 NOVEMBER 1971 NUMBER11 [CANCER RESEARCH 31, 1523-1542, November 1971] A Review of Isozymes in Cancer1 Wayne E. Criss Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida 32601 TABLE OF CONTENTS postulated role for that particular isozymic system in cellular metabolism. Summary 1523 Introduction 1523 Normal enzyme differentiation 1523 INTRODUCTION Tumor enzyme differentiation 1524 Isozymes 1524 Normal Enzyme Differentiation DNA polymerase 1524 Enzyme differentiation is the process whereby, during the Hexokinase 1525 Fructose 1,6-diphosphatase 1525 development of an organ in an animal, the organ acquires the quantitative and qualitative adult enzyme patterns (122). Key Aldolase 1526 pathway enzymes in several metabolic processes have been Pyruvate kinase 1527 found to undergo enzymatic differentiation. The enzymes Láclatedehydrogenase 1527 Isocitrate dehydrogenase 1527 involved in nitrogen metabolism, and also in urea cycle Malate dehydrogenase 1528 metabolism (180), are tyrosine aminotransferase (123, 151, Glycerol phosphate dehydrogenase 1529 330, 410), tryptophan pyrrolase (261), serine dehydratase Glutaminase 1529 (123, 410), histidine ammonia lyase (11), and aspartate Aspartate aminotransferase 1530 aminotransferase (337, 388). The enzymes involved in nucleic Adenylate kinase 1531 acid metabolism are DNA polymerase (156, 277) and RNase (52). In glycolysis the enzymes are hexokinase-glucokinase Carbamyl phosphate synthetase 1531 Lactose synthetase 1533 (98, 389), galactokinase 30, aldolase (267, 315), pyruvate Discussion 1533 kinase (73, 386), and lactate dehydrogenase (67, 69). In References 1533 mitochondrial oxidation they are NADH oxidase, succinic oxidase, a-glycero-P oxidase, ATPase, cytochrome oxidase, and flavin content (84, 296). In glycogen metabolism the SUMMARY enzymes involved are UDPG pyrophosphorylase and UDPG glucosyltransferase (19).
    [Show full text]
  • A Novel DNA Primase-Helicase Pair Encoded by Sccmec Elements Aleksandra Bebel†, Melissa a Walsh, Ignacio Mir-Sanchis‡, Phoebe a Rice*
    RESEARCH ARTICLE A novel DNA primase-helicase pair encoded by SCCmec elements Aleksandra Bebel†, Melissa A Walsh, Ignacio Mir-Sanchis‡, Phoebe A Rice* Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States Abstract Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase- polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3’-to-5’ helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec. *For correspondence: Introduction [email protected] Staphylococcus aureus is a dangerous human pathogen, due in part to the emergence of multi- drug-resistant strains such as MRSA (methicillin-resistant S. aureus). MRSA strains have acquired † Present address: Phage resistance to b-lactam antibiotics (including methicillin) mainly through horizontal gene transfer of a Consultants, Gdynia, Poland; mobile genomic island called staphylococcal cassette chromosome (SCC) (Moellering, 2012). ‡Umea˚ University, Umea˚ , SCCmec is a variant of SCC that carries a methicillin resistance gene, mecA.
    [Show full text]