DNA Synthesis Guided Discussion
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Glossary - Cellbiology
1 Glossary - Cellbiology Blotting: (Blot Analysis) Widely used biochemical technique for detecting the presence of specific macromolecules (proteins, mRNAs, or DNA sequences) in a mixture. A sample first is separated on an agarose or polyacrylamide gel usually under denaturing conditions; the separated components are transferred (blotting) to a nitrocellulose sheet, which is exposed to a radiolabeled molecule that specifically binds to the macromolecule of interest, and then subjected to autoradiography. Northern B.: mRNAs are detected with a complementary DNA; Southern B.: DNA restriction fragments are detected with complementary nucleotide sequences; Western B.: Proteins are detected by specific antibodies. Cell: The fundamental unit of living organisms. Cells are bounded by a lipid-containing plasma membrane, containing the central nucleus, and the cytoplasm. Cells are generally capable of independent reproduction. More complex cells like Eukaryotes have various compartments (organelles) where special tasks essential for the survival of the cell take place. Cytoplasm: Viscous contents of a cell that are contained within the plasma membrane but, in eukaryotic cells, outside the nucleus. The part of the cytoplasm not contained in any organelle is called the Cytosol. Cytoskeleton: (Gk. ) Three dimensional network of fibrous elements, allowing precisely regulated movements of cell parts, transport organelles, and help to maintain a cell’s shape. • Actin filament: (Microfilaments) Ubiquitous eukaryotic cytoskeletal proteins (one end is attached to the cell-cortex) of two “twisted“ actin monomers; are important in the structural support and movement of cells. Each actin filament (F-actin) consists of two strands of globular subunits (G-Actin) wrapped around each other to form a polarized unit (high ionic cytoplasm lead to the formation of AF, whereas low ion-concentration disassembles AF). -
Paul Modrich Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA
Mechanisms in E. coli and Human Mismatch Repair Nobel Lecture, December 8, 2015 by Paul Modrich Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA. he idea that mismatched base pairs occur in cells and that such lesions trig- T ger their own repair was suggested 50 years ago by Robin Holliday in the context of genetic recombination [1]. Breakage and rejoining of DNA helices was known to occur during this process [2], with precision of rejoining attributed to formation of a heteroduplex joint, a region of helix where the two strands are derived from the diferent recombining partners. Holliday pointed out that if this heteroduplex region should span a genetic diference between the two DNAs, then it will contain one or more mismatched base pairs. He invoked processing of such mismatches to explain the recombination-associated phenomenon of gene conversion [1], noting that “If there are enzymes which can repair points of damage in DNA, it would seem possible that the same enzymes could recognize the abnormality of base pairing, and by exchange reactions rectify this.” Direct evidence that mismatches provoke a repair reaction was provided by bacterial transformation experiments [3–5], and our interest in this efect was prompted by the Escherichia coli (E. coli) work done in Matt Meselson’s lab at Harvard. Using artifcially constructed heteroduplex DNAs containing multiple mismatched base pairs, Wagner and Meselson [6] demonstrated that mismatches elicit a repair reaction upon introduction into the E. coli cell. Tey also showed that closely spaced mismatches, mismatches separated by a 1000 base pairs or so, are usually repaired on the same DNA strand. -
Chromosome Replication Duringmeiosis
Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3087-3091, November 1973 Chromosome Replication During Meiosis: Identification of Gene Functions Required for Premeiotic DNA Synthesis (yeast) ROBERT ROTH Biology Department, Illinois Institute of Technology, Chicago, Ill. 60616 Communicated by Herschel L. Roman, May 29, 1973 ABSTRACT Recent comparisons of chromosome repli- tained provide additional evidence that distinct biochemical cation in meiotic and mitotic cells have revealed signifi- reactions do distinguish the last premeiotic replication from cant differences in both the rate and pattern of DNA synthesis during the final duplication preceding meiosis. replication during growth. These differences suggested that unique gene functions might be required for premeiotic replication that were not MATERIALS AND METHODS necessary for replication during growth. To provide Yeast Strains. Mutants M10-2B and M10-6A were isolated evidence for such functions, we isolated stage-specific mutants in the yeast Saccharomyces cerevisiae which per- from disomic (n + 1) strain Z4521-3C. The original disome mitted vegetative replication but blocked the round of used to construct Z4521-3C was provided by Dr. G. Fink (13). replication before meiosis. The mutants synthesized car- Construction and properties of Z4521-3C and details of mu- bohydrate, protein, and RNA during the expected interval tant isolation have been described (12). Z4521-3C and both of premeiotic replication, suggesting that their lesions preferentially affected synthesis of DNA. The mutations mutants have the following general structure: blocked meiosis, as judged by a coincident inhibition of intragenic recombination and ascospore formation. The leu2-27 a lesions were characterized as recessive nuclear genes, and + + + ade2-1, met2, ura3 his 4 leu2- + a (III) were designated mei-1, mei-2, and mei-3; complementa- ade-1,met, ua3his 4 leu 2-1 aa thr 4 tion indicated that the relevant gene products were not p identical. -
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. -
Studies on in Vitro DNA Synthesis.* Purification of the Dna G Gene
Proc. Nat. Acad. Sci. USA Vol. 70, No. 5, pp. 1613-1618, May 1973 Studies on In Vitro DNA Synthesis.* Purification of the dna G Gene Product from Escherichia coli (dna A, dna B, dna C, dna D, and dna E gene products/+X174/DNA replication/DNA polymerase III) SUE WICKNER, MICHEL WRIGHT, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Alfred Gilman, March 12, 1973 ABSTRACT q5X174 DNA-dependent dNMP incorpora- Hirota; BT1029, (polA1, thy, endo I, dna B ts) and BT1040 tion is temperature-sensitive (ts) in extracts of uninfected endo I, thy, dna E ts), isolated by F. Bonhoeffer and E. coli dna A, B, C, D, E, and G ts strains. DNA synthesis (polAi, can be restored in heat-inactivated extracts of various dna co-workers and obtained from J. Wechsler; PC22 (polA1, his, ts mutants by addition of extracts of wild-type or other strr, arg, mtl, dna C2 ts) and PC79 (polAi, his, star, mtl, dna D7 dna ts mutants. A protein that restores activity to heat- ts), derivatives (4) of strains isolated by P. L. Carl (3) and inactivated extracts of dna G ts cells has been extensively obtained from M. Gefter. DNA was prepared by the purified. This protein has also been purified from dna G ts OX174 cells and is thermolabile when compared to the wild-type method of Sinsheimer (15) or Franke and Ray (16). protein. The purified dna G protein has a molecular weight of about 60,000, is insensitive to N-ethylmaleimide, and Preparation of Receptor Crude Extracts. -
Inducers of DNA Synthesis Present During Mitosis of Mammalian Cells Lacking G1 and G2 Phases (Cell Cycle/Cell Fusion/Prematurely Condensed Chromosomes) POTU N
Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 5043-5047, October 1978 Cell Biology Inducers of DNA synthesis present during mitosis of mammalian cells lacking G1 and G2 phases (cell cycle/cell fusion/prematurely condensed chromosomes) POTU N. RAO, BARBARA A. WILSON, AND PRASAD S. SUNKARA Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Communicated by David M. Prescott, July 27, 1978 ABSTRACT The cell cycle analysis of Chinese hamster lung MATERIALS AND METHODS fibroblast V79-8 line by the premature chromosome condensa- tion method has confirmed the absence of measurable GI and Cells and Cell Synchrony. The Chinese hamster cell line G2 periods. Sendai virus-mediated fusion of mitotic V79-8 cells (V79-8), which lacks both the GI and G2 phases in its cell cycle, with GI phase HeLa cells resulted in the induction of both DNA was kindly supplied by R. Michael Liskay, University of Col- synthesis and premature chromosome condensation in the latter, orado, Boulder, CO. V79-8 cells were grown as monolayers on indicating the presence of the inducers of DNA synthesis above Falcon plastic culture dishes in McCoy's 5A modified medium the critical level not only throughout S phase, as it is in HeLa, supplemented with 15% heat-inactivated fetal calf serum but also during mitosis of V79-8 cells. No initiation of DNA (GIBCO) in a humidified CO2 (5%) incubator at 37°. Under synthesis was observed whe-n GI phase HeLa cells were fused these conditions, this cell line had a generation time of about with mitotic CHO cells. -
Control of Eukaryotic DNA Replication Initiation—Mechanisms to Ensure Smooth Transitions
G C A T T A C G G C A T genes Review Control of Eukaryotic DNA Replication Initiation—Mechanisms to Ensure Smooth Transitions Karl-Uwe Reusswig and Boris Pfander * Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, 82152 Martinsried, Germany; [email protected] * Correspondence: [email protected] Received: 31 December 2018; Accepted: 25 January 2019; Published: 29 January 2019 Abstract: DNA replication differs from most other processes in biology in that any error will irreversibly change the nature of the cellular progeny. DNA replication initiation, therefore, is exquisitely controlled. Deregulation of this control can result in over-replication characterized by repeated initiation events at the same replication origin. Over-replication induces DNA damage and causes genomic instability. The principal mechanism counteracting over-replication in eukaryotes is a division of replication initiation into two steps—licensing and firing—which are temporally separated and occur at distinct cell cycle phases. Here, we review this temporal replication control with a specific focus on mechanisms ensuring the faultless transition between licensing and firing phases. Keywords: DNA replication; DNA replication initiation; cell cycle; post-translational protein modification; protein degradation; cell cycle transitions 1. Introduction DNA replication control occurs with exceptional accuracy to keep genetic information stable over as many as 1016 cell divisions (estimations based on [1]) during, for example, an average human lifespan. A fundamental part of the DNA replication control system is dedicated to ensure that the genome is replicated exactly once per cell cycle. If this control falters, deregulated replication initiation occurs, which leads to parts of the genome becoming replicated more than once per cell cycle (reviewed in [2–4]). -
De Novo DNA Synthesis Using Polymerase- Nucleotide Conjugates
De novo DNA synthesis using polymerase- nucleotide conjugates Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des Grades Doktor rerum naturalium (Dr. rer. nat) Dissertation von Sebastian Palluk Erstgutachterin: Prof. Dr. Beatrix Süß Zweitgutachter: Prof. Dr. Johannes Kabisch Darmstadt 2018 Palluk, Sebastian: De novo DNA synthesis using polymerase-nucleotide conjugates Darmstadt, Technische Universität Darmstadt Jahr der Veröffentlichung der Dissertation: 2019 Tag der mündlichen Prüfung: 17.12.2018 Veröffentlicht unter CC BY-NC-SA 4.0 International https://creativecommons.org/licenses/ 2 Summary The terminal deoxynucleotidyl transferase (TdT) is the key enzyme proposed for enzy- matic DNA synthesis, based on its ability to extend single stranded DNA rapidly using all four different deoxynucleoside triphosphates (dNTPs). Proposals to employ TdT for the de novo synthesis of defined DNA sequences date back to at least 1962, and typically involve using the polymerase together with 3’-modified reversible terminator dNTPS (RTdNTPs), analogous to Sequencing by Synthesis (SBS) schemes. However, polymerases usually show a low tolerance for 3’-modified RTdNTPs, and the catalytic site of TdT seems particularly difficult to engineer in order to enable fast incorporation kinetics for such modified dNTPs. Until today, no practical enzymatic DNA synthesis method based on this strategy has been published. Here, we developed a novel approach to achieve single nucleotide extension of a DNA molecule by a polymerase. By tethering a single dNTP to the polymerase in a way that it can be incorporated by the polymerase moiety, we generate so called polymerase-nucleotide conjugates. Once a polymerase-nucleotide conjugate extends a DNA molecule by its tethered dNTP, the polymerase moiety stays covalently attached to the extended DNA via the linkage to the incorporated nucleotide, and blocks other polymerase-nucleotide conjugates from accessing the 3’-end of the DNA molecule. -
DNA Replication
DNA replication • DNA replication • Process of Replication in E. coli • Origin of replication • Role of Primase: RNA Primer • Elongation • Lagging strand synthesis: Okazaki fragments • Error rate of DNA synthesis • Eukaryotic Replication Types of replication Meselson-Stahl Experiment: Semi-conservative replication Eukaryotic chromosomes with base Analog 5-Bromodeoxyuridine with staining Process of Replication in E. coli Polymerases • DNA Polymerases • I,II, III • No initiation of replication • Primase: RNA polymerase • Lays down RNA nucleotides (primer) Origin of replication • Origin: 245 bp, containing repeats • Proteins involved, DNA A (initial denaturing), DNA B and C (further opening/destabilize helix) • unwinding of the helix: helicases (DNA B/C) • stabilization of the helix: single stranded binding proteins • role of topoisomerases, DNA gyrases Initiation Elongation • Anti-parallel strands • DNA Polymerase III • Leading strand synthesis DNA polymerase Replisome Lagging strand synthesis • Role of DNA Polymerase I • removal of primer • exonuclease activity • DNA ligase DNA Ligase Proofreading • Error rate of DNA synthesis • Proofreading • Base Pairing rules Eukayotic Replication • Multiple origins • Polymerases • Linear chromosomes Multiple origins Eukaryotic DNA Replication • DNA helicase promotes unwinding at the replication fork, • DNA pol δ with RFC and PCNA synthesizes DNA on the leading strand. • DNA pol α initiates synthesis on the lagging strand by generating an RNA primer (red segment) followed by a short segment of DNA. Then, RFC and PCNA load a second DNA polymerase (δ or ε ) to continue synthesis of the Okazaki fragment. • B, as DNA pol δ approaches the downstream Okazaki fragment, • Cleavage by RNase H1 removes the initiator RNA primer leaving a single 5 -ribonucleotide. Then, FEN1/RTH1 removes the 5 -ribonucleotide. -
De Novo DNA Synthesis Using Polymerase
LETTERS De novo DNA synthesis using polymerase- nucleotide conjugates Sebastian Palluk1–3,12, Daniel H Arlow1,2,4,5,12, Tristan de Rond1,2,6, Sebastian Barthel1–3, Justine S Kang1,2,7, Rathin Bector1,2,7, Hratch M Baghdassarian1,2,8, Alisa N Truong1,2, Peter W Kim1,9, Anup K Singh1,9, Nathan J Hillson1,2,10 & Jay D Keasling1,2,5,7,8,11 Oligonucleotides are almost exclusively synthesized using the oligos in a process that is failure-prone and not amenable to all target nucleoside phosphoramidite method, even though it is limited sequences10, rendering some DNA sequences inaccessible to study. to the direct synthesis of ~200 mers and produces hazardous Proposals for enzymatic de novo synthesis of oligonucleotides with waste. Here, we describe an oligonucleotide synthesis strategy a defined sequence date back to at least 1962 (refs. 11,12). Enzymatic that uses the template-independent polymerase terminal oligo synthesis promises several potential advantages over chemical deoxynucleotidyl transferase (TdT). Each TdT molecule is synthesis: 1) the exquisite specificity of enzymes and mild conditions conjugated to a single deoxyribonucleoside triphosphate in which they function may reduce the formation of side products (dNTP) molecule that it can incorporate into a primer. After and DNA damage such as depurination, thereby enabling the direct incorporation of the tethered dNTP, the 3′ end of the primer synthesis of longer oligos; 2) reactions take place in aqueous condi- remains covalently bound to TdT and is inaccessible to other tions and need not generate hazardous waste; 3) synthesis could be TdT–dNTP molecules. Cleaving the linkage between TdT and initiated from natural DNA (i.e., DNA without protecting groups on the incorporated nucleotide releases the primer and allows the nucleophilic positions of the bases); and 4) enzyme engineering subsequent extension. -
Helicase-DNA Polymerase Interaction Is Critical to Initiate Leading-Strand DNA Synthesis
Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis Huidong Zhang1, Seung-Joo Lee1, Bin Zhu, Ngoc Q. Tran, Stanley Tabor, and Charles C. Richardson2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, April 27, 2011 (sent for review March 3, 2011) Interactions between gene 4 helicase and gene 5 DNA polymerase (gp5) are crucial for leading-strand DNA synthesis mediated by the replisome of bacteriophage T7. Interactions between the two pro- teins that assure high processivity are known but the interactions essential to initiate the leading-strand DNA synthesis remain uni- dentified. Replacement of solution-exposed basic residues (K587, K589, R590, and R591) located on the front surface of gp5 with neu- tral asparagines abolishes the ability of gp5 and the helicase to mediate strand-displacement synthesis. This front basic patch in gp5 contributes to physical interactions with the acidic C-terminal tail of the helicase. Nonetheless, the altered polymerase is able to replace gp5 and continue ongoing strand-displacement synthesis. The results suggest that the interaction between the C-terminal tail of the helicase and the basic patch of gp5 is critical for initiation of strand-displacement synthesis. Multiple interactions of T7 DNA polymerase and helicase coordinate replisome movement. DNA polymerase-helicase interaction ∣ strand-displacement DNA synthesis ∣ T7 bacteriophage ∣ T7 replisome acteriophage T7 has a simple and efficient DNA replication Bsystem whose basic reactions mimic those of more complex replication systems (1). The T7 replisome consists of gene 5 DNA polymerase (gp5), the processivity factor, Escherichia coli Fig. -
Part 2 - Modeling DNA Function: Replication Questions to Jumpstart Your Thinking 1
...where molecules become real TM DYNAMIC DNA KIT© © Student Guide DYNAMIC DNA KIT Part 2 - Modeling DNA Function: Replication Questions to jumpstart your thinking 1. Why do cells divide? 2. One of the most common kitchen injuries is cuts from kitchen equipment. When someone sustains such an injury, how do original cells compare to the new cells once the wound has healed? 3. How does your body ensure that new cells for growth and repair contain the same information? 4. How does DNA get into the new cells? Introduction The first famousNature paper published by James Watson and Francis Crick in April of 1953, entitled “Molecular Structure of Nucleic Acids,” ends with the quote, “It has not escaped our notice that the specific pairing we have postulated immediately suggest a possible copying mechanism for the genetic material.” In a second paper published in Nature in May of 1953, entitled “Genetical Implications of the Structure of Deoxyribonucleic Acid,” Watson and Crick suggest a mechanism for how DNA replicates: “Now our model for deoxyribonucleic acid is, in effect, a pair of templates, each of which is complementary to the other. We imagine that prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then acts as a template for the formation on to itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before. Moreover, the sequence of the pairs of bases will have been duplicated exactly.” Since then, many scientists have focused on researching the mechanism of DNA replication.