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

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Chapter 25 DNA Metabolism Replication, Repair and Recombination

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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


1
Chapter 25 DNA Metabolism Replication, Repair and
Recombination
  • 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)

2
E. Coli grown on 15N first Transferred to 14N
medium Samples withdrawn at different
generations Subjected to CsCl gradient
centrifugation
3
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

4
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

5
Chain Elongation Is a Nucleotidyl-Group-Transfer
Reaction
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
    strand
  • 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

6
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

7
Bacteriophage DNA polymerase bound to DNA
Proofreading Corrects Polymerization Errors
8
DNA Polymerase Synthesizes Two Strands
Simultaneously
9
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)
10
DNA polymerase I activities
  • The 5 3' activity of DNA pol I removes the
    RNA primer at the beginning of each Okazaki
    fragment
  • Synthesizes nick translation polymerase
    activity synthesizes DNA in place of RNA

Nick Break in the DNA backbone
11
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

12
3 Stages of DNA Replication in E. coli
  • 1. Initiation 2. Elongation 3.
    Termination
  • 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

13
(No Transcript)
14
  • Elongation
  • DNA helicases unwinding ahead of fork
  • SSBs stabilize single strands
  • Primase synthesizes RNA primers
  • 1 for leading strand
  • 1 for each Okazaki fragment

15
  • Core catalyzes polymerization reaction
  • ? subunits clamp for processivity
  • Lagging strand
  • - ? subunits load template onto ? clamp
  • - New one every Okazaki fragment

16
  • 1000 nucleotides/s
  • Process completed by DNA pol I and DNA ligase

17
Termination
  • 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

18
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

19
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
    SSB
  • Helicases also present to unwind DNA
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