DOCSLIB.ORG

Stability of the Human Polymerase Δ Holoenzyme and Its Implications In

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Stability of the Human Polymerase Δ Holoenzyme and Its Implications In Stability of the human polymerase δ holoenzyme and PNAS PLUS its implications in lagging strand DNA synthesis Mark Hedglina, Binod Pandeya, and Stephen J. Benkovica,1 aDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802 Edited by Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved February 17, 2016 (received for review November 30, 2015) In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicat- the front face of the clamp is oriented toward the 3′ end of the ing the lagging strand template and anchors to the proliferating cell nascent primer/template (P/T) junction where DNA synthesis nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The will initiate. An incoming pol δ subsequently captures the stability of this complex is integral to every aspect of lagging strand loaded PCNA ring, forming a holoenzyme, and DNA synthesis replication. Most of our understanding comes from Saccharomyces initiates (2, 5). cerevisae where the extreme stability of the pol δ holoenzyme en- The stability of the lagging strand holoenzyme is integral to sures that every nucleobase within an Okazaki fragment is faithfully various aspects of Okazaki fragment synthesis. For eukaryotes, duplicated before dissociation but also necessitates an active dis- most of our understanding comes from studies in Saccharomyces placement mechanism for polymerase recycling and exchange. cerevisae, where the three-subunit pol δ is extremely stable with However, the stability of the human pol δ holoenzyme is unknown. −3 −1 PCNA on DNA (koff < 2 × 10 s , t1/2 > 5 min). Once DNA We designed unique kinetic assays to analyze the processivity and synthesis is initiated from a nascent primer, the dramatically slow stability of the pol δ holoenzyme. Surprisingly, the results indicate k ensures every nucleotide within a given Okazaki fragment is that human pol δ maintains a loose association with PCNA while off faithfully duplicated before dissociation. On the other hand, such replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on high stability necessitates an active mechanism for displacement of δ pol δ once DNA synthesis stops (6, 7). This situation arises when a undamaged DNA and promotes the rapid dissociation of pol from δ ′ PCNA on stalling at a DNA lesion. pol holoenzyme encounters either the 5 RNA end of a down- stream Okazaki fragment (pol recycling) or distortions to the BIOCHEMISTRY lagging strand | stability | PCNA | DNA polymerase delta | native sequence that it cannot accommodate (pol exchange), such translesion DNA synthesis as common byproducts of UV radiation exposure (8). Pol recy- cling allows the scarce pol δ to be reused during S-phase, whereas uring S-phase of the cell cycle, genomic DNA must be pol exchange permits a specialized pol to bind to PCNA and faithfully copied in a short period. Replicative DNA poly- synthesize past the offending damage [translesion DNA syn- D δ – merases (pols) alone are distributive and must anchor to ring- thesis (TLS)] so that pol may resume synthesis (9 12). How- shaped sliding clamps to achieve the high degree of processivity ever, studies on the human pol δ holoenzyme are lacking, and required for efficient DNA replication. The highly conserved to- hence, the mechanisms by which polymerase recycling and ex- roidal structure of sliding clamps has a central cavity large enough change occur are unknown. To gain insight, we designed a to encircle double-stranded DNA (dsDNA) and slide freely along unique kinetic assay to measure the stability of the pol δ ho- it. Thus, such an association effectively tethers the pol to DNA, loenzyme. Surprisingly, the results indicate that human pol δ substantially increasing the extent of continuous replication. The maintains a loose association with PCNA. Such behavior has eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), profound implications on lagging strand synthesis as it limits is trimer of identical subunits aligned head-to-tail, forming a ring the extent of processive DNA synthesis and promotes the rapid with two structurally distinct faces. Each subunit consists of two dissociation of pol δ from PCNA on stalling. independent domains connected by an interdomain connecting “ ” loop (IDCL). The front face of the homotrimeric PCNA ring Significance contains all IDCLs and is a platform for interaction with the eukaryotic replicative pols, e and δ, which are responsible for the The results from the reported studies indicate that the human faithful replication of the leading and lagging strands, respec- lagging strand polymerase, pol δ, maintains a loose association tively (1, 2). Specifically, the well-conserved PCNA-interacting with the sliding clamp, proliferating cell nuclear antigen (PCNA), peptide (PIP) box within replicative pols makes extensive contact while replicating and rapidly dissociates on stalling, leaving PCNA with an IDCL of PCNA and displays conserved residues that behind on the DNA. This behavior has profound implications on “plug” into the proximal hydrophobic patches. The amino acid lagging strand synthesis as it limits the extent of processive DNA sequence of a canonical PIP box is QXXhXXaa, where X rep- synthesis on undamaged DNA and promotes the rapid dissociation resents any amino acid, h is a hydrophobic residue (usually L, I, of pol δ on stalling at a replication-blocking lesion. This challenges or M), and a is an aromatic residue (usually F or Y) (3). the accepted models for polymerase recycling and exchange on the Unlike the leading strand, the lagging strand is synthesized lagging strand and instead suggests passive mechanisms for the discontinuously in short Okazaki fragments that are processed human system. These studies provide valuable insight for future and ligated together to form a continuous strand (4). In eu- experiments in the fields DNA replication, DNA repair, and DNA karyotes, each Okazaki fragment is initiated by the bifunctional damage tolerance. DNA pol α/primase complex that lays down cRNA/DNA hybrid – primers every 100 250 nucleotides (nt) on the exposed tem- Author contributions: M.H. and S.J.B. designed research; M.H. and B.P. performed re- plate for the lagging strand. The intermittent single-stranded search; M.H., B.P., and S.J.B. analyzed data; and M.H. and S.J.B. wrote the paper. DNA (ssDNA) is protected from cellular nucleases by repli- The authors declare no conflict of interest. cation protein A (RPA), a ssDNA binding protein that also This article is a PNAS Direct Submission. prevents formation of alternative DNA structures. The clamp 1To whom correspondence should be addressed. Email: [email protected]. loader, replication factor C (RFC), recognizes these hybrid This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. primers abutted by RPA and loads PCNA onto each such that 1073/pnas.1523653113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1523653113 PNAS Early Edition | 1of10 Downloaded by guest on September 23, 2021 amino acid (13–21). The homotrimeric PCNA sliding clamp contains three identical binding sites for PIP box-containing pro- teins and, hence, human pol δ may simultaneously bind all three subunits within a given PCNA trimer, similar to that observed in S. cerevisae (22). Indeed, sequential removal of the p12 and p66 Fig. 1. Sequence of the biotinylated primer/template P29/Bio62. The size of accessory subunits from the human pol δ assembly reduced the the double-stranded P/T region (29 bp) is in agreement with the size of an initiating hybrid P/T and the requirements for assembly of a human pol δ extent of PCNA-dependent DNA synthesis in a stepwise manner, holoenzyme by RFC (5, 70). The biotin attached to the 3′-end of the template suggesting that all PCNA-interacting subunits are required to form strand was prebound to neutravidin, preventing loaded PCNA from sliding off a holoenzyme with optimal DNA synthetic activity (23). In all the 5′ end of the primer. The single-stranded DNA (ssDNA) adjacent to the 3′ experiments discussed herein, only the complete, four-subunit end of the P/T junction was prebound with excess RPA. The size of this region human pol δ complex was used. Furthermore, pol exchange in (33 nt) is consistent with the size of ssDNA covered by a single RPA molecule eukaryotes involves the conjugation of single ubiquitin moieties – δ (22 30 nt) (71). To monitor extension of the primer by pol within a single (i.e., monoubiquitination) to lysine residues (K164) of PCNA re- binding encounter, the 29-mer primer was 32P-end-labeled before annealing and reactions were carried out in the presence of a passive DNA trap. In all siding at a stalled P/T junction. This posttranslational modification experiments, the trap is unlabeled substrate lacking a biotin tag and con- (PTM) is essential for optimal TLS activity in mammalian cells taining a 3′-dideoxy-terminated primer (referred to as 29ddC/62). (24), but its role in pol exchange is under intense debate. To gain insight into a potential role of this PTM involving the lagging strand pol δ, we synthesized monoubiquitinated PCNA [referred Results to herein as (Ub)3-PCNA] that contains a single ubiquitin moiety Monitoring a Single Turnover of Primer Extension by Human pol δ. on K164 of each monomer within a homotrimeric clamp ring (25). Human pol δ is comprised of four subunits: three accessory sub- The conjugation of ubiquitin to PCNA has no effect on the in- units (p50/POLD2, p66/POLD3, and p12/POLD4) and a large teraction of PCNA with RFC (Fig. S1B) or on the ability of RFC catalytic subunit (p125/POLD1) that contains both DNA poly- to load PCNA onto DNA (Fig. S2), in agreement with observa- merase and exonuclease domains.
Load more

Recommended publications
  • Teacher Notes Science Nspired
    DNA Replication TEACHER NOTES SCIENCE NSPIRED Science Objectives In this lesson, students will • Explore how the structure of DNA supports its semi-conservative replication • Identify the name and function of several key enzymes in DNA replication • Recognize the function of Okazaki fragments in DNA replication Vocabulary TI-Nspire™ Technology Skills: • Double helix • Leading strand • Download a TI-Nspire • Lagging strand • Polymerase I • Polymerase III • Helicase document • Base pairs • Okazaki Fragments • Open a document • Primase • Move between pages • Open Directions Box About the Lesson • Explore ‘Hot Spots’ • Using three simulations, students will interact with DNA replication to explore semi-conservative replication and identify Tech Tips: specific enzymes and their roles in replication. Assessments are Make sure that students know embedded in the activity to engage discussion and gauge how to move between pages by learning. pressing /¡ (left arrow) and • As a result, students will: /¢ (right arrow). • Learn the basic functions of the following DNA replication enzymes: helicase, primase, ligase, polymerase I and III. Lesson Materials: • Learn how the double helix reproduces in a semi- Student Activity • DNA_Replication_Student.do conservative fashion c • Learn the role and function of Okazaki fragments in • DNA_Replication_Student.pd replication of the lagging strand. f TI-Nspire document TI-Nspire™ Navigator™ • DNA_Replication.tns Not required. If using Navigator: • Send out the DNA_Replication.tns file. • Monitor student progress
    [Show full text]
  • DNA Structure and Replication Three Key Features Needed for Any Model
    DNA structure and replication What was known? 1) Hereditary factors were associated with specific traits 2) One-gene-one-protein model - from mapping genes for biosynthetic pathways 3) Genes are on chromosomes 4) Chromosomes are made up of DNA and protein Three key features needed for any model of DNA structure 1) Must allow for faithful replication 2) Must have information content 3) Must be able to change in order to explain mutations 1 Rosalind Franklin James Watson Francis Crick X-ray diffraction Maurice Wilkins Photograph of B-DNA Linus Pauling Nature 171, 737-738 (1953) © Macmillan Publishers Ltd. Molecular structure of Nucleic Acids WATSON, J. D. & CRICK, F. H. C. Features of the double helix A Structure for Deoxyribose Nucleic Acid 1) Two parallel strands We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest………… 2) Bases held together by H-bonds 3) Phosphodiester backbone 4) The base is attached to the position 1 on the sugar 5) Base pair stack - provides Figure 1. This figure is purely diagrammatic. The two ribbons symbolize the two phophate-sugar chains, and the horizonal rods stability the pairs of bases holding the chains together. The vertical line marks the fibre axis. 6) Contains a major and …………….It has not escaped our notice that the specific pairing we minor groove have postulated immediately suggests a possible copying mechanism for the genetic material. Three key features needed for any model of DNA structure
    [Show full text]
  • Processivity of DNA Polymerases: Two Mechanisms, One Goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2
    Minireview 121 Processivity of DNA polymerases: two mechanisms, one goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2 Replicative DNA polymerases are highly processive Processive DNA synthesis by cellular replicases and the enzymes that polymerize thousands of nucleotides without bacteriophage T4 replicase dissociating from the DNA template. The recently Until recently, the only mechanism for high processivity determined structure of the Escherichia coli bacteriophage that was understood in detail was that utilized by cellular T7 DNA polymerase suggests a unique mechanism that replicases and the replicase of bacteriophage T4. This underlies processivity, and this mechanism may generalize mechanism involves a ring-shaped protein called a ‘DNA to other replicative polymerases. sliding clamp’ that encircles the DNA and tethers the polymerase catalytic unit to the DNA [3,4]. The three- Addresses: 1Department of Molecular Biology, Memorial Sloan- dimensional structures of several sliding clamps have been Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, 2 determined: the eukaryotic proliferating cell nuclear USA and Laboratory of DNA Replication, Howard Hughes Medical β Institute, The Rockefeller University, 1230 York Avenue, New York, NY antigen (PCNA) [5,6]; the subunit of the prokaryotic 10021, USA. DNA polymerase III [7]; and the bacteriophage T4 gene 45 protein (gp45) (J Kuriyan, personal communication) *Corresponding author. (Figure 1). The overall structure of these clamps is very E-mail: [email protected] similar; the PCNA, β subunit and gp45 rings are super- Structure 15 February 1998, 6:121–125 imposable [8]. Each ring has similar dimensions and a http://biomednet.com/elecref/0969212600600121 central cavity large enough to accommodate duplex DNA (Figure 1).
    [Show full text]
  • Primer Release Is the Rate-Limiting Event in Lagging-Strand Synthesis
    Primer release is the rate-limiting event in lagging- strand synthesis mediated by the T7 replisome Alfredo J. Hernandeza, Seung-Joo Leea, and Charles C. Richardsona,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, April 18, 2016 (sent for review December 30, 2015; reviewed by Nicholas E. Dixon and I. Robert Lehman) DNA replication occurs semidiscontinuously due to the antiparallel but also for enabling the use of short oligoribonucleotides by T7 DNA strands and polarity of enzymatic DNA synthesis. Although DNA polymerase. Critically, the primase domain also fulfills two the leading strand is synthesized continuously, the lagging strand additional roles apart from primer synthesis: it prevents disso- is synthesized in small segments designated Okazaki fragments. ciation of the extremely short tetramer, stabilizing it with the Lagging-strand synthesis is a complex event requiring repeated template, and it secures it in the polymerase active site (10, 12). cycles of RNA primer synthesis, transfer to the lagging-strand Here we show that the rate-limiting step in initiation of Okazaki polymerase, and extension effected by cooperation between DNA fragments by the T7 replisome is primer release from the primase primase and the lagging-strand polymerase. We examined events domain of gp4. In the absence of gp2.5, an additional step, distinct controlling Okazaki fragment initiation using the bacteriophage from primer release, also limits primer extension. The presence of T7 replication system. Primer utilization by T7 DNA polymerase is gp2.5 promotes efficient primer formation and primer utilization. slower than primer formation.
    [Show full text]
  • Junction Ribonuclease: an Activity in Okazaki Fragment Processing
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2244–2249, March 1998 Biochemistry Junction ribonuclease: An activity in Okazaki fragment processing RICHARD S. MURANTE,LEIGH A. HENRICKSEN, AND ROBERT A. BAMBARA* Department of Biochemistry and Biophysics, and Cancer Center, Box 712, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642 Communicated by I. Robert Lehman, Stanford University School of Medicine, Stanford, CA, December 29, 1997 (received for review September 15, 1997) ABSTRACT The initiator RNAs of mammalian Okazaki and removal of RNA primers. Reconstitution assays with both fragments are thought to be removed by RNase HI and the mouse cell and SV40 replication proteins require RNase HI for 5*-3* flap endonuclease (FEN1). Earlier evidence indicated the maturation of lagging strands in vitro (12, 13). that the cleavage site of RNase HI is 5* of the last ribonucle- Although cleavage of long RNAyDNA hybrids is random, otide at the RNA-DNA junction on an Okazaki substrate. In cleavage of certain substrates is clearly structure specific. Eder current work, highly purified calf RNase HI makes this exact et al. (16) found that when a series of ribonucleotides were cleavage in Okazaki fragments containing mismatches that followed by a 39 DNA segment and annealed to DNA to form distort the hybrid structure of the heteroduplex. Furthermore, a duplex, human RNase HI preferentially cleaved to leave a even fully unannealed Okazaki fragments were cleaved. DNA segment with a 59 monoribonucleotide. In reactions Clearly, the enzyme recognizes the transition from RNA to reconstituting Okazaki fragment processing, RNase HI from DNA on a single-stranded substrate and not the RNAyDNA calf thymus similarly cleaved the RNA-DNA strand 59 to the heteroduplex structure.
    [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]
  • Plasmid Replication-Associated Single-Strand-Specific
    12858–12873 Nucleic Acids Research, 2020, Vol. 48, No. 22 Published online 3 December 2020 doi: 10.1093/nar/gkaa1163 Plasmid replication-associated single-strand-specific methyltransferases Alexey Fomenkov 1,*, Zhiyi Sun1, Iain A. Murray 1, Cristian Ruse1, Colleen McClung1, Yoshiharu Yamaichi 2, Elisabeth A. Raleigh 1,* and Richard J. Roberts1,* 1New England Biolabs Inc., 240 County Road, Ipswich, MA, USA and 2Universite´ Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Downloaded from https://academic.oup.com/nar/article/48/22/12858/6018438 by guest on 24 September 2021 Received August 06, 2020; Revised November 10, 2020; Editorial Decision November 11, 2020; Accepted November 12, 2020 ABSTRACT RM-associated modification and the diversity of associ- ated functions remains incompletely understood (3). Long- Analysis of genomic DNA from pathogenic strains read, modification-sensitive SMRT sequencing technology of Burkholderia cenocepacia J2315 and Escherichia has facilitated sequencing and assembly of a wide variety coli O104:H4 revealed the presence of two unusual of genomes and also clarified the modification repertoire. MTase genes. Both are plasmid-borne ORFs, carried Detection of the modification status of bases is possible by pBCA072 for B. cenocepacia J2315 and pESBL for for m6A, m4C and oxidized forms of m5C modified bases E. coli O104:H4. Pacific Biosciences SMRT sequenc- (m5hC and 5caC) (4). Recently, high throughput analy- ing was used to investigate DNA methyltransferases sis of 230 diverse bacterial and archaeal methylomes strik- M.BceJIII and M.EcoGIX, using artificial constructs. ingly revealed that almost 50% of organisms harbor Type Mating properties of engineered pESBL derivatives II DNA methyltransferases (MTase) homologs with no ap- were also investigated.
    [Show full text]
  • Review Questions DNA Replication
    Review Questions DNA Replication 1. Explain semi-conservative replication. Prior to cell division, a cell must make a copy of its DNA to pass along to the next generation. Copying DNA is called “replication”. Rather than build a DNA molecule from scratch, the new DNA is composed of one old DNA strand (used as the template) and one brand new strand. “Semi-conservative” means that half of the new DNA molecule is old DNA. 2. How can the speed of DNA replication increase while the rate of replication remains constant? The conundrum of DNA replication is that in humans the replication enzymes can copy at a rate of 50 base pairs per second. That may seem like a fast rate but there are 3.1 billion base pairs in the human genome. At that rate, if the machinery started at one end of the DNA and replicated all the way down to the other end, it would take ~ 2 years to copy one DNA molecule. Replication occurs much faster than that. How? Well, the answer is that DNA replication starts at many places along the molecule. These separate “origins of replication” form “replication bubbles”. Once a bubble forms, replication moves in both directions. These expanding bubbles of replication will eventually meet and the whole genome will be copied in a matter of hours rather than years. 3. Explain the process of DNA replication. Unwinding and Unzipping the Double Helix. The two strands in a DNA molecule are connected by hydrogen bonds between the complementary bases. An enzyme called “helicase” travels along the DNA unwinding and breaking the hydrogen bonds between the two strands.
    [Show full text]
  • Bt6504 – Molecular Biology Question Bank
    JEPPIAAR ENGINEERING COLLEGE B.TECH – BIOTECHNOLOGY (R- 2013) BT6504 – MOLECULAR BIOLOGY III YEAR & V SEM BATCH: 2016-2020 QUESTION BANK PREPARED BY Mr. G. GOMATHI SANKAR G. Gomathi Sankar S.N TOPIC REFER PAGE O ENCE NO UNIT I – CHEMISTRY OF NUCLEIC ACIDS 1 Introduction to nucleic acids TB1 79 2 Nucleic acids as genetic material TB1 79 3 Structure and physicochemical properties of elements in DNA and RNA TB1 80-82 4 Biological significance of differences in DNA and RNA TB1 84-92 5 Primary structure of DNA: Chemical and structural qualities of 3’,5’- TB1 94-96 Phosphodiester bond 6 Secondary Structure of DNA: Watson & Crick model TB1 97-100 7 Chargaff’s rule TB1 109 8 X–ray diffraction analysis of DNA, Forces stabilizes DNA structure TB1 110-112 9 Conformational variants of double helical DNA, Hogsteen base pairing TB1 114 10 Triple helix, Quadruple helix, Reversible denaturation and hyperchromic effect TB1 112-114 11 Tertiary structure of DNA: DNA supercoiling. TB1 148 UNIT II – DNA REPLICATION & REPAIR 1 Overview of Central dogma TB1 208 2 Organization of prokaryotic and eukaryotic chromosomes DNA replication: TB1 209 Meselson & Stahl experiment, bi–directional DNA replication 3 Okazaki fragments TB1 210 4 Proteomics of DNA replication, Fidelity of DNA replication TB1 224-231 5 Inhibitors of DNA replication TB1 232-235 6 Overview of differences in prokaryotic and eukaryotic DNA replication TB1 271-273 7 Telomere replication in eukaryotes TB1 239-246 8 D-loop and rolling circle mode of replication TB1 255-258 9 Mutagens, DNA mutations
    [Show full text]
  • DNA Replication
    DNA replication: • Copying genetic information for transmission to the next generation • Occurs in S phase of cell cycle • Process of DNA duplicating itself • Begins with the unwinding of the double helix to expose the bases in each strand of DNA • Each unpaired nucleotide will attract a complementary nucleotide from the medium – will form base pairing via hydrogen bonding. • Enzymes link the aligned nucleotides by phosphodiester bonds to form a continuous strand. 1 DNA replication: – First question asked was whether duplication was semiconservative or conservative • Meselson and Stahl expt • Semiconservative - – one strand from parent in each new strand • Conservative- – both strands from parent and other is all new strands 2 DNA replication: • Complementary base pairing produces semiconservative replication – Double helix unwinds – Each strand acts as template – Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C – Two daughter helices produced after replication 3 4 Experimental proof of semiconservative replication – three possible models • Semiconservative replication – – Watson and Crick model • Conservative replication: – The parental double helix remains intact; – both strands of the daughter double helix are newly synthesized • Dispersive replication: – At completion, both strands of both double helices contain both original and newly synthesized material. 5 6 Meselson-Stahl experiments confirm semiconservative replication • Experiment allowed differentiation of parental and newly formed DNA. • Bacteria were grown in media containing either normal isotope of nitrogen (14N) or the heavy isotope (15N). • DNA banded after equilibrium density gradient centrifugation at a position which matched the density of the DNA: – heavy DNA was at a higher density than normal DNA.
    [Show full text]
  • Single-Molecule Analysis Reveals That the Lagging Strand Increases Replisome Processivity but Slows Replication Fork Progression
    Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression Nina Y. Yao1, Roxana E. Georgescu1, Jeff Finkelstein, and Michael E. O’Donnell2 Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by Michael E. O’Donnell, June 3, 2009 (sent for review May 14, 2009) Single-molecule techniques are developed to examine mechanistic The current report examines the rate and processivity of the features of individual E. coli replisomes during synthesis of long E. coli replisome. The E. coli replisome consists of the ring DNA molecules. We find that single replisomes exhibit constant shaped hexameric DnaB helicase, DnaG primase, the DNA rates of fork movement, but the rates of different replisomes vary polymerase III* replicase (Pol III*), and ␤ sliding clamps. over a surprisingly wide range. Interestingly, lagging strand syn- Processivity estimates of the E. coli replisome from ensemble thesis decreases the rate of the leading strand, suggesting that studies indicate a lower limit of 50 kb, and an average bulk rate lagging strand operations exert a drag on replication fork progres- of Ϸ500–700 nt/s at 37 °C (6, 7). Processivity measurements from sion. The opposite is true for processivity. The lagging strand other recent single-molecule studies differ over a wide range, significantly increases the processivity of the replisome, possibly from3kbtoϾ80 kb (8, 9). In each of these previous studies reflecting the increased grip to DNA provided by 2 DNA poly- replication was performed in the presence of excess proteins that merases anchored to sliding clamps on both the leading and could replace a replisome protein that dissociates from DNA, lagging strands.
    [Show full text]
  • DNA Replication in Prokaryotes
    392 Chapter 14 | DNA Structure and Function 14.4 | DNA Replication in Prokaryotes By the end of this section, you will be able to do the following: • Explain the process of DNA replication in prokaryotes • Discuss the role of different enzymes and proteins in supporting this process DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes. DNA replication employs a large number of structural proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain.
    [Show full text]