DOCSLIB.ORG

Herpes Simplex Virus 1 Helicase-Primase: a Complex of Three Herpes-Encoded Gene Products (Replication Origin/DNA Binding Protein/DNA Polymerase) JAMES J

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Herpes Simplex Virus 1 Helicase-Primase: a Complex of Three Herpes-Encoded Gene Products (Replication Origin/DNA Binding Protein/DNA Polymerase) JAMES J Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2186-2189, April 1989 Biochemistry Herpes simplex virus 1 helicase-primase: A complex of three herpes-encoded gene products (replication origin/DNA binding protein/DNA polymerase) JAMES J. CRUTE*, TATSUYA TsURUMI*, LIANG ZHUt, SANDRA K. WELLERt, PAUL D. OLIVOt, MARK D. CHALLBERGt, EDWARD S. MOCARSKI§, AND I. R. LEHMAN* Departments of *Biochemistry and §Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; tDepartment of Microbiology, University of Connecticut Health Center, Farmington, CT 06032; and tLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Bethesda, MD 20892 Contributed by I. R. Lehman, January 3, 1989 ABSTRACT In an earlier report, we described a DNA Cells and Viruses. A305, a thymidine kinase-deficient helicase that is specifically induced upon infection ofVero cells mutant of HSV-1[F], was used to infect roller-bottle cultures with herpes simplex virus 1. We have purified this enzyme to of Vero cells at a multiplicity of infection of 5 plaque-forming near homogeneity and found it to consist of three polypeptides units per cell. with molecular weights of 120,000, 97,000, and 70,000. Im- Buffers. The buffers used were described (14) except that munochemical analysis has shown these polypeptides to be the the concentration of glycerol in buffer A was increased to products of three of the genes UL52, UL5, and UL8 that are 20% (vol/vol) and the concentrations of leupeptin and pep- required for replication of a plasmid containing a herpes statin A were increased to 10 ,ug/ml. simplex 1 origin (oris). In addition to helicase activity, the Enzymatic Assays. DNA-dependent ATPase and GTPase enzyme contains a tightly associated DNA primase. Thus, the assays were performed as described (14) except that incuba- three-subunit enzyme is a helicase-primase complex that may tion was for 20 min at 34TC. DNA primase assays were carried prime lagging-strand synthesis as it unwinds DNA at the viral out essentially as described (15). The reaction mixture, in 30 replication fork. A.l, contained 50 mM Tris HCl (pH 8.7), 3.5 mM MgCI2, 10% (vol/vol) glycerol, bovine serum albumin at 100 jig/ml, 1.0 mM ATP, 13 ,uM [3H]dATP (10 Ci/mmol; 1 Ci = 37 GBq), 40 The 153-kilobase genome of herpes simplex virus 1 (HSV-1) ,uM (dT)6", and 1.0 unit ofthe large fragment ofEscherichia contains both cis- and trans-acting elements that function in coli DNA polymerase I obtained from United States Bio- viral DNA replication (1). The cis-acting elements corre- chemical. The reaction was started by the addition of 3.0 ,ul spond to the origins of DNA replication (oris and oriL) (2-4), of enzyme solution and, after 60 min at 34°C, was stopped by and the trans-acting elements very likely code for most and the addition of 10 ,ul of 0.5 M EDTA. Incorporation of possibly all of the enzymes required for HSV-1 DNA repli- [3H]dATP into acid-insoluble material was determined as cation. The nucleotide sequence ofthe entire HSV-1 genome described (16). One unit of primase activity incorporated 1.0 has been determined (5), allowing assignment ofHSV-1 genes pmol of [3H]dATP in 60 min. Nuclease assays were per- and their products to specific open reading frames. Seven of formed as described (14). these open reading frames have been shown to be necessary Gel Electrophoresis and Immunoblot Analysis. SDS/poly- and sufficient for the replication in trans of plasmids con- acrylamide gel electrophoresis was performed and the gels taining either origin of DNA replication, oriL or oris (6). were silver-stained as described (17, 18). Immunoblot anal- These open reading frames also correspond to seven com- yses were performed (19, 20) using rabbit antiserum against plementation groups known to be essential for HSV-1 DNA the UL5 gene product expressed in E. coli (21) or rabbit replication (7-9). Of the seven open reading frames, three antisera raised against peptides whose sequence corresponds have thus far been identified and shown to encode the herpes to the predicted carboxyl-terminal 10 amino acid residues of DNA polymerase (Pol) (10), a single-stranded DNA-binding the HSV-1 UL8 or UL52 open reading frames (5, 22). protein (ICP8) (11), and the oris-binding protein (UL9) (12). Preparation of Nuclei. HSV-1-infected Vero cells were A double-stranded DNA-binding protein whose role in DNA harvested from roller bottles 14-15 hr after infection by replication is unknown is encoded by the fourth open reading vigorous shaking. The nuclei were prepared by the hypotonic frame (UL42) (13). lysis method (23), except that the nuclear wash was omitted In this report we show that the HSV-1-induced DNA and the concentrations of leupeptin and pepstatin A were helicase that we have identified (14) consists of three poly- increased to 10 ,g/ml. peptides encoded by the three remaining open reading frames Enzyme Purification. The HSV-1 DNA helicase was puri- UL5, UL8, and UL52. We have also found that a DNA fied as described (14) with several modifications. In immu- primase activity is tightly associated with the three-subunit noblotting experiments with UL5 antibody, we had found enzyme, establishing the presence of an HSV-1-encoded that the UL5 gene product corresponded to the HSV- helicase-primase complex in HSV-1-infected cells. 1-induced helicase, as assayed by its DNA-dependent ATPase (GTPase) activity (J.J.C., L.Z., S.K.W., and I.R.L., unpublished results). We also observed that the amount of MATERIALS AND METHODS DNA-dependent ATPase as well as the level of intact UL5 Materials. Reagents and substrates have been described gene product varied considerably among our nuclear prepa- (14), unless otherwise noted. (dT)6w0 was obtained from rations, very likely as a result of uncontrolled proteolysis. To Midland Certified Reagent (Midland, TX). ensure that nuclei containing undegraded helicase were used for enzyme purification, we immunoblotted small amounts (100 ,ul) of the high-salt nuclear extract as a means of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: HSV-1, herpes simplex virus 1. 2186 Downloaded by guest on September 28, 2021 Biochemistry: Crute et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2187 screening for full-length UL5 gene product. Of five nuclear mally active at pH 8.5-9.0 and at Mg2+ concentrations from preparations tested, two were found suitable for further 1 to 3 mM. processing; the remainder showed considerable degradation. HSV-1 Helicase-Primase Is Composed of Three Subunits. Nuclei (60 g) containing intact UL5 product were thawed and Purification of the DEAE-Sephadex fraction by filtration centrifuged, and the pellets were resuspended in an equal through Superose 12 again yielded a single peak of DNA- volume of buffer A supplemented with 90 mM NaCI. One- dependent ATPase that coincided with primase activity. The eighth volume of buffer A containing 4.0 M NaCl was added step also removed a small amount of contaminating nuclease and the nuclear suspension was centrifuged at 90,000 rpm for (Fig. 2A). In agreement with the previous report (14), the 10 min at 20C in a TLA.100.3 rotor. The supernatant solution molecular weight of the helicase-primase complex was was dialyzed against buffer A containing 50 mM NaCl until -440,000. The specific activity of the primase was 2.2 X 104 the NaCl concentration was 100 mM. The extract was units/mg and that of the DNA-dependent ATPase was 2.5 X chromatographed on phosphocellulose and DEAE-Seph- 104 units/mg. The DNA-dependent ATPase and primase adex, and a portion (40%) of the DEAE-Sephadex fraction activities also coincided with a single peak of protein as was filtered through a Superose 12 column as described (14). measured by absorbance at 280 nm. When fractions across Approximately 20 jig of near-homogeneous enzyme was the peak of helicase and primase activity were analyzed by recovered. SDS/polyacrylamide gel electrophoresis, three polypeptides with molecular weights of 120,000, 97,000, and 70,000 were RESULTS found to increase and decrease concomitant with the two enzymatic activities as well as with the peak of protein (Fig. Association of Primase Activity with HSV-1 Helicase. The 2B). As estimated by silver staining, the three polypeptides HSV-1-induced DNA helicase, assayed by its DNA- accounted for >95% of the protein in the peak. dependent ATPase (GTPase) activity, can be effectively Identification ofthe Subunits ofthe HSV-1 Helicase-Primase separated from host DNA-dependent ATPase activity by as the Products of UL52, UL5, and UL8 Genes. We sought to chromatography on phosphocellulose (14). Further purifica- identify which, if any, of the three polypeptides associated tion of the herpes enzyme by chromatography on DEAE- with the helicase-primase were encoded by the seven HSV-1 Sephadex yielded two fractions, one that failed to adsorb to genes that are required for origin-dependent DNA replica- the column and a second that did adsorb and subsequently tion. The UL52, UL5, and UL8 genes, which encode proteins could be eluted (14). There was, however, considerable with predicted molecular weights of 114,000, 99,000, and variability in the relative amounts of the two fractions, and, 80,000, respectively, were the most likely candidates (6). We indeed, in a number ofpreparations, activity was not retained therefore immunoblotted fractions across the Superose 12 by the column at all.
Load more

Recommended publications
  • Saccharomyces Rrm3p, a 5 to 3 DNA Helicase That Promotes Replication
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Saccharomyces Rrm3p, a 5؅ to 3؅ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA Andreas S. Ivessa,1 Jin-Qiu Zhou,1,2 Vince P. Schulz, Ellen K. Monson, and Virginia A. Zakian3 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric ؅ C1–3A/TG1–3 tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5 ,to 3؅ DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere at the natural X-bearing Chromosome III-L telomere, and at Y؅-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C1–3A/TG1–3 DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication. [Key Words: Telomere; helicase; telomerase; replication; RRM3; yeast] Received February 7, 2002; revised version accepted April 10, 2002. Telomeres are the natural ends of eukaryotic chromo- Because conventional DNA polymerases cannot repli- somes. In most organisms, the very ends of chromo- cate the very ends of linear DNA molecules, special somes consist of simple repeated sequences. For ex- mechanisms are required to prevent the loss of terminal ample, Saccharomyces cerevisiae chromosomes end in DNA.
    [Show full text]
  • The Architecture of a Eukaryotic Replisome
    The Architecture of a Eukaryotic Replisome Jingchuan Sun1,2, Yi Shi3, Roxana E. Georgescu3,4, Zuanning Yuan1,2, Brian T. Chait3, Huilin Li*1,2, Michael E. O’Donnell*3,4 1 Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA 2 Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York, USA. 3 The Rockefeller University, 1230 York Avenue, New York, New York, USA. 4 Howard Hughes Medical Institute *Correspondence and requests for materials should be addressed to M.O.D. ([email protected]) or H.L. ([email protected]) ABSTRACT At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase leads the way in front to unwind DNA, and that DNA polymerases (Pol) trail behind the helicase. Here we use single particle electron microscopy to directly image a replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, while Ctf4 and the lagging strand Pol α-primase (Pol α) are behind the helicase. This unexpected architecture indicates that the leading strand DNA travels a long distance before reaching Pol ε, it first threads through the Mcm2-7 ring, then makes a U-turn at the bottom to reach Pol ε at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture, and provides a basis for further structural and biochemical replisome studies. INTRODUCTION DNA is replicated by a multi-protein machinery referred to as a replisome 1,2. Replisomes contain a helicase to unwind DNA, DNA polymerases that synthesize the leading and lagging strands, and a primase that makes short primed sites to initiate DNA synthesis on both strands.
    [Show full text]
  • The Biochemical Activities of the Saccharomyces Cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain
    G C A T T A C G G C A T genes Article The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain David G. Nickens y, Christopher W. Sausen y and Matthew L. Bochman * Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA; [email protected] (D.G.N.); [email protected] (C.W.S.) * Correspondence: [email protected] These authors contributed equally to this work. y Received: 31 March 2019; Accepted: 20 May 2019; Published: 28 May 2019 Abstract: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1DN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1DN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity. Keywords: DNA helicase; Saccharomyces cerevisiae; Pif1; telomerase; telomere 1. Introduction DNA helicases are enzymes that couple DNA binding and ATP hydrolysis to unwind double-stranded DNA (dsDNA) into its component single strands [1].
    [Show full text]
  • Exploring the Non-Canonical Functions of Metabolic Enzymes Peiwei Huangyang1,2 and M
    © 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm033365. doi:10.1242/dmm.033365 REVIEW SPECIAL COLLECTION: CANCER METABOLISM Hidden features: exploring the non-canonical functions of metabolic enzymes Peiwei Huangyang1,2 and M. Celeste Simon1,3,* ABSTRACT A key finding from studies of metabolic enzymes is the existence The study of cellular metabolism has been rigorously revisited over the of mechanistic links between their nuclear localization and the past decade, especially in the field of cancer research, revealing new regulation of transcription. By modulating gene expression, insights that expand our understanding of malignancy. Among these metabolic enzymes themselves facilitate adaptation to rapidly insights isthe discovery that various metabolic enzymes have surprising changing environments. Furthermore, they can directly shape a ’ activities outside of their established metabolic roles, including in cell s epigenetic landscape (Kaelin and McKnight, 2013). the regulation of gene expression, DNA damage repair, cell cycle Strikingly, several metabolic enzymes exert completely distinct progression and apoptosis. Many of these newly identified functions are functions in different cellular compartments. Nuclear fructose activated in response to growth factor signaling, nutrient and oxygen bisphosphate aldolase, for example, directly interacts with RNA ́ availability, and external stress. As such, multifaceted enzymes directly polymerase III to control transcription (Ciesla et al., 2014),
    [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]
  • Mcm10 Has Potent Strand-Annealing Activity and Limits Translocase-Mediated Fork Regression
    Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression Ryan Maylea, Lance Langstona,b, Kelly R. Molloyc, Dan Zhanga, Brian T. Chaitc,1,2, and Michael E. O’Donnella,b,1,2 aLaboratory of DNA Replication, The Rockefeller University, New York, NY 10065; bHoward Hughes Medical Institute, The Rockefeller University, New York, NY 10065; and cLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 Contributed by Michael E. O’Donnell, November 19, 2018 (sent for review November 8, 2018; reviewed by Zvi Kelman and R. Stephen Lloyd) The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, of function using genetics, cell biology, and cell extracts have GINS) tightly binds Mcm10, an essential replication protein in all identified Mcm10 functions in replisome stability, fork progres- eukaryotes. Here we show that Mcm10 has a potent strand- sion, and DNA repair (21–25). Despite significant advances in the annealing activity both alone and in complex with CMG. CMG- understanding of Mcm10’s functions, mechanistic in vitro studies Mcm10 unwinds and then reanneals single strands soon after they of Mcm10 in replisome and repair reactions are lacking. have been unwound in vitro. Given the DNA damage and replisome The present study demonstrates that Mcm10 on its own rap- instability associated with loss of Mcm10 function, we examined the idly anneals cDNA strands even in the presence of the single- effect of Mcm10 on fork regression. Fork regression requires the strand (ss) DNA-binding protein RPA, a property previously unwinding and pairing of newly synthesized strands, performed by associated with the recombination protein Rad52 (26).
    [Show full text]
  • Huh7 HK4+ HK2- Cells a Protein Complementation Assay B Coimmunoprecipitation Even If NS3 Is Able to Stimulates Glycolysis in Cells Expressing Replicate
    Dengue virus protein NS3 activates hexokinase activity in SAT-390 hepatocytes to support virus replication Marianne FIGL, Clémence JACQUEMIN, Patrice ANDRE, Laure PERRIN-COCON, Vincent LOTTEAU, Olivier DIAZ International Center for Infectiology Research (CIRI), INSERM U1111, CNRS UMR5308, Université de Lyon, FRANCE 1 INTRODUCTION 4 RESULTS 5 CONCLUSIONS Result 2: DENV NS3 protein interacts with hexokinases Viruses are mandatory parasites that use metabolism machinery to Result 1: DENV efficiently replicates in HuH7 HK4+ HK2- cells A Protein Complementation Assay B Coimmunoprecipitation Even if NS3 is able to stimulates glycolysis in cells expressing replicate. Growing literature demonstrates that viruses manipulate A.A. DENV-NS3 versus human metabolism enzymes DENVB.B.-NS3 versus hexokinases A. HuH7 HuH7 HK4+ HK2- B. (a) (b) 60 Lysate Co-IP HK2 or HK4, we observe an higher DENV replication in HuH7 central carbon metabolism (CCM) and more specifically glycolysis for HuH7 HuH7 55 NS3-3xFlag - + - + HuH7 HK4+ HK2- HK4+ HK2- suggesting that HK4 positive cells are more susceptible their propagation [1]. However, the underlying mechanisms are not HuH7 HK4+ HK2- 50 HK1 α-Gluc 45 α-Flag to DENV replication. fully described. Our team has already demonstrated that hepatitis C 40 80 80 HK2 α-Gluc *** 35 NS5A protein interacts and activates hexokinases (HKs) to favor viral 70 70 α-Flag cells 30 Poster presented at: presented Poster Fluorescente light Fluorescente 60 cells 60 α-Gluc replication [2]. It was described that dengue infection (DENV) 25 HK3 We observed that HuH7 HK4+HK2- cells have a rewiring of their 50 50 α-Flag 20 increases glycolysis [3] and thus we wondered if control of 40 40 glycolytic pathway resulting in intracellular lipids accumulation (see 15 HK4 α-Gluc 30 hexokinase activity was shared by DENV, another Flavivirus.
    [Show full text]
  • Managing DNA Polymerases: Coordinating DNA Replication, DNA Repair, and DNA Recombination
    Colloquium Managing DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombination Mark D. Sutton and Graham C. Walker* Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 Two important and timely questions with respect to DNA replica- A Superfamily of DNA Polymerases Involved in Replication of Imper- tion, DNA recombination, and DNA repair are: (i) what controls fect DNA Templates. Recently, the field of translesion DNA which DNA polymerase gains access to a particular primer-termi- synthesis and induced mutagenesis has generated a great deal of nus, and (ii) what determines whether a DNA polymerase hands off excitement because of the discovery that key gene products its DNA substrate to either a different DNA polymerase or to a required for these processes, in both prokaryotes (9, 10) and in different protein(s) for the completion of the specific biological eukaryotes (11, 12), possess an intrinsic DNA polymerase ac- process? These questions have taken on added importance in light tivity (refs. 6, 7, and 13–20 and reviewed in refs. 21–24). A of the fact that the number of known template-dependent DNA common, defining feature of these DNA polymerases is a polymerases in both eukaryotes and in prokaryotes has grown remarkable ability to replicate imperfect DNA templates. De- tremendously in the past two years. Most notably, the current list pending on the DNA polymerase, these include templates such now includes a completely new family of enzymes that are capable as those containing a misaligned primer–template junction (13), of replicating imperfect DNA templates. This UmuC-DinB-Rad30- an abasic site (6, 7), a cyclobutane dimer (15, 16, 25), or a pyrimidine–pyrimidone (6–4) photoproduct (25).
    [Show full text]
  • Key for Exam 3 • Biology II • Winter 2013 Multiple Choice Questions
    Key for Exam 3 • Biology II • Winter 2013 Multiple Choice Questions. Circle the one best answer for each question. (1 point each) 1. Which of the following is not part of the cell theory: A. All cells come from other cells. B. Only eukaryotic cells have membrane-bounded organelles. C. The cell is the smallest living unit of a living thing. D. All living things are made up of cells. 2. A protein that belongs in the plasma membrane of a eukaryotic cell might be found (at some point) in which organelle: A. Golgi apparatus B. ribosome C. cytoplasmic reticulum D. nucleus E. mitochondrion 3. A phospholipid is composed of: A. three fatty acids and a molecule of glycerol B. chains of hydrophobic amino acids C. two fatty acids, a molecule of glycerol and a highly hydrophilic group D. amphipathic fatty acids E. a phosphate, a ribose or deoxyribose sugar, and a fatty acid 4. A membrane would be more permeable if: A. …its phospholipids had longer tails. B. …its phospholipids had shorter tails. C. …it contained more cholesterol. D. …its phospholipids had more saturated tails. E. …its proteins were more hydrophobic. 5. An enzyme is working at its Vmax: A. …at the high point of the product vs. time curve. B. …when its active site is continuously full of substrate. C. …when the concentration of substrate is equal to its km. D. …when it is assayed at its optimum temperature and pH. E. …at the early time points when its slope is the steepest. 6. In an enzyme-catalyzed reaction, the role of the enzyme is to: A.
    [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]
  • Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene
    Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1995 Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene. Haiqing Fu Schiltz Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Schiltz, Haiqing Fu, "Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene." (1995). LSU Historical Dissertations and Theses. 6075. https://digitalcommons.lsu.edu/gradschool_disstheses/6075 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. Hie quality of this reproduction Is dependent upon the quality of the copy snbmitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandardm a rg in * , and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are mi«ing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g^ maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and confirming from left to right in equal sections with small overlaps.
    [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]