Chapter 25 DNA Metabolism Replication, Repair and Recombination - PowerPoint PPT Presentation


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Chapter 25 DNA Metabolism Replication, Repair and Recombination


Each strand of DNA acts as a ... synthesis of one strand DNA polymerase II - role in DNA repair DNA ... DNA in place of RNA Nick Break in the ... – PowerPoint PPT presentation

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Title: Chapter 25 DNA Metabolism Replication, Repair and Recombination

Chapter 25 DNA Metabolism Replication, Repair and
  • Semiconservative DNA replication
  • Each strand of DNA acts as a template for
    synthesis of a new strand
  • Daughter DNA contains one parental and one newly
    synthesized strand

Meselson-Stahl Experiment
  • 1953 Watson-Crick Structure
  • 1957 This experiment proves the semi-conservative
    model of DNA replication
  • Nitrogen-15 isotope used (heavy)
  • Nitrogen-14 isotope (most common, light)

E. Coli grown on 15N first Transferred to 14N
medium Samples withdrawn at different
generations Subjected to CsCl gradient
Chromosomal DNA Replication is Bidirectional
  • E. coli chromosome is circular, double-stranded
    DNA (4.6x103 kilobase pairs)
  • Replication begins at a unique site (origin)
  • Proceeds bidirectionally until the two
    replication complexes meet (termination site)
  • Replisome - protein machinery for replication
    (one replisome at each of 2 replication forks)
  • Duplication in about 38 minutes
  • Bidirectional DNA replication in E. coli
  • New strands of DNA are synthesized at the two
    replication forks where replisomes are located

Eukaryotic replication
  • Eukaryotic chromosomes are large linear,
    double-stranded DNA molecules
  • Fruit fly large chromosomes 5.0x104 kb (10x
    larger than E. coli)
  • Replication is also bidirectional
  • Multiple sites of initiation of DNA synthesis
    (versus one site in E. coli)

Replicating DNA in the fruit fly
  • Large number of replication forks at opposite
    ends of bubbles of duplicated DNA

Chain Elongation Is a Nucleotidyl-Group-Transfer
DNA Polymerase
  • E. coli contains at least 5 DNA polymerases
  • DNA polymerase I - repairs DNA and participates
    in DNA synthesis of one strand
  • DNA polymerase II - role in DNA repair
  • DNA polymerase III - the major DNA replication
    enzyme, responsible for chain elongation
  • Base pair between incoming deoxynucleotide 5'
    triphosphate (blue) and a residue of the parental
  • Terminal 3' OH attacks a-phosphorous of incoming
    nucleotide to form new phosphodiester linkage
  • Strand growth always in the 5' ? 3' direction
  • Irreversible due to formation or PPi which is
    quickly hydrolyzed to 2Pi by pyrophosphatase

DNA PolIII Remains Bound to the Replication Fork
  • DNA polymerase III is a processive enzyme
    (remains bound to the replication fork until
    replication is complete)
  • Certain subunits form a sliding clamp which
    surrounds the DNA molecule
  • Two ?-subunits associate to form a head-to-tail
    dimer in the shape of a ring that completely
    surrounds the DNA
  • Remaining subunits of DNA pol III are bound to
    this structure

Bacteriophage DNA polymerase bound to DNA
Proofreading Corrects Polymerization Errors
DNA Polymerase Synthesizes Two Strands
RNA Primer Begins Each New Strand, including
each Okazaki Fragment
  • Primosome is a complex containing primase enzyme
    which synthesizes short pieces of RNA at the
    replication fork (complementary to the
    lagging-strand template)
  • DNA pol III uses the RNA primer to start the
    lagging-strand DNA synthesis
  • Replisome - includes primosome, DNA pol III
  • Okazaki fragments are joined to produce a
    continuous strand of DNA in 3 steps

(1) Removal of the RNA primer (pol I) (2)
Synthesis of replacement DNA (pol I) (3) Sealing
of adjacent DNA fragments (DNA ligase)
DNA polymerase I activities
  • The 5 3' activity of DNA pol I removes the
    RNA primer at the beginning of each Okazaki
  • Synthesizes nick translation polymerase
    activity synthesizes DNA in place of RNA

Nick Break in the DNA backbone
DNA ligase activity
  • Klenow (large) fragment of DNA pol I, lacks 5'?3'
    exonuclease activity
  • Used for DNA synthesis
  • Catalyzes the formation of a phosphodiester
    linkage between 3-hydroxyl and 5-phosphate of
    adjacent Okazaki fragments
  • Eukaryotic enzymes require ATP cosubstrate
  • E. coli DNA ligase uses NAD as a cosubstrate

Model of the Replisome
  • Replisome contains a primosome, DNA polymerase
    III holoenzyme, additional proteins
  • DnaB helicase is part of the primosome and
    facilitates unwinding of the DNA helix
  • Topoisomerases relieve supercoiling ahead of the
    replicating fork (not part of the replisome)
  • Single-stranded binding proteins (SSBs) stabilize
    single-stranded DNA

3 Stages of DNA Replication in E. coli
  • 1. Initiation 2. Elongation 3.
  • Initiation
  • Regulated for once per cell cycle
  • Replisome assembles at origin site (oriC)
  • Origin site is a highly conserved sequence and
    contains two series of short repeats
  • DnaA is first initiation protein
  • binds at four 9 bp repeat sequences
  • causes denaturation at three 13 bp repeats
  • requires ATP and HU (histone-like protein)
  • Hexamers of DnaB (aided by DnaC) unwind DNA

(No Transcript)
  • Elongation
  • DNA helicases unwinding ahead of fork
  • SSBs stabilize single strands
  • Primase synthesizes RNA primers
  • 1 for leading strand
  • 1 for each Okazaki fragment

  • Core catalyzes polymerization reaction
  • ? subunits clamp for processivity
  • Lagging strand
  • - ? subunits load template onto ? clamp
  • - New one every Okazaki fragment

  • 1000 nucleotides/s
  • Process completed by DNA pol I and DNA ligase

  • Terminator utilization substance (Tus) binds to
    the ter site
  • Tus inhibits helicase activity and thus prevents
    replication forks continuing through this region
  • ter sites act as replication block
  • Last few hundred bp made by unknown mechanism
  • Topoisomerase IV frees catenated DNAs
  • Catenanes - circles wound around each other

DNA Replication in Eukaryotes
  • Mechanism similar to that in prokaryotes leading
    strand continuous synthesis, lagging strand
    discontinuous synthesis
  • Replication forks move more slowly, but many
    replication forks (50 nucleotides/s)
  • Okazaki fragments are shorter in eukaryotes
    (100-200 residues)
  • At least 5 different DNA polymerases

Accessory proteins associated with the
replication fork
  • PCNA (proliferating cell nuclear antigen) forms
    structure resembling ?-subunit sliding clamp (E.
    coli DNA polymerase III)
  • RPC (replication factor C) similar to ? complex
    of DNA pol III
  • RPA (replication factor A) similar to prokaryotic
  • Helicases also present to unwind DNA