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Genetics of Respiratory Disease

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Genetics of Respiratory Disease Powered By Docstoc
					                 DNA Replication

                   Dr. Jiandong Huang
                Department of Biochemistry
               The University of Hong Kong



January 2009
   Questions About Replication
• How does replication begin, how does it progress
  along the chromosome,
• How does replication terminated, and what
  mechanism ensure that only one round of
  replication occurs before cell division?
• Which enzymes take part in DNA replication, and
  what are their functions?
• How does duplication of the long helical duplex
  occur without the strands being tangled?
 Overview of DNA Replication
• General Features
   –   Time of DNA replication
   –   Semiconservative
   –   Replication origin
   –   Bidirectional
• DNA replication machinery
   –   Initiation proteins
   –   Helicase
   –   Primase
   –   Polymerases
   –   Leading and lagging strands
   –   Telomerase
• Topoisomerases in DNA replication
    Is DNA Replication Dispersive,
   Conservative or Semiconservative?
• All models
  fit the
  Watson-
  Crick model
  of DNA
  prediction:
  exact
  copying of
  genetic
  information.
  Equilibrium
    Density
Centrifugation in
 DNA Analysis



 M. Meselson
 W. F. Stahl
Semiconservative Replication of Density-Labeled DNA
      Is DNA Replication Unidirectional
             or Bidirectional?
• (a). Linear DNA
  virus
• (b). Some bacterial
  plasmid
• (c). Most organisms
Evidence for
Bidirectional
Replication
Evidence for Bidirectional Replication
 Summary of the Common Features
    Chromosomal Replication

• Replication Origin
• Semiconservative
• Bidirectional
    Replication of Bacterial DNA from a Single Origin




Single replication origin in bacteria.
Multiple Replicons in Eukaryotic
             DNA
• Replicon: each region
  served by one origin.
• Several thousand
  replicon in large
  eukaryotic cells.
• 50-300 kb each
The Replication Bubble in
  Drosophila embryos
            Replication Origin
• DNA replication starts from specific
  chromosomal sites




    Origins of replication in E. coli
       9-mers (dnaA box)
       AT rich 13-mers
  Bacterial Replication and Cell division




No apparent chromosome
condensation
Origin used to separate
chromosomes
Yeast Origin: Autonomously
Replicating Sequences (ARS)
Common Features of Replication
          Origin
• Unique DNA sequence containing multiple
  short repeated sequence
• Recognized by multimeric origin-binding
  proteins
• AT-rich stretch
    Rate of DNA replication
• E. coli
   –   42 minutes
   –   4,639,221 bp
   –   1.4 mm
   –   1000 bp/second/fork
• Human
   –   8 hours
   –   3 x 109 bp
   –   2m
   –   100 bp/second/fork
   –   10,000 to 100,000 replicons
DNA Replication Machinery
          DNA Polymerase
• Unable to separate the two strands of DNA
• Only elongate a pre-existing DNA or RNA
  (Primer)
• Only add nucleotides to the 3’-hyforxyl
  group, i.e., only 5’-3’ synthesis
     Genes and Proteins in DNA
             Repliction
•   Gene        Protein
•   dnaA        DnaA
•   dnaB        DnaB or Helicase
•   dnaC        DnaC
•   dnaG        Primase
 Initiation of DNA Replication
• DnaA Protein Initiates Replication in E. coli
   – Initiation complex
   – Prepriming complex
        Initiation of DNA Replication
• DnaB is a helicase.
   – An enzyme moves
     along DNA duplex
     utilizing the energy of
     ATP hydrolysis to
     separate the strands.
   – 5’-3’
   – Processive
• SSB: single strand
  binding protein.
The Role of RNA Primer
  in DNA Replication


   • E. coli primase
     catalyze the RNA
     Primer for DNA
     Synthesis
     – dnaG
   • Primosome:
     primases+DnaB
   • Primer: <15 nts
   DNA Pol III
• 600 kDa
• Holoemzyme: 10
  peptides
• Core polymerase:
    α: active site
  – ε: 3’-5’ exonuclease
  – θ: unkown
    β-subunit dimer tethers the core of
   E. coli DNA polymerase III to DNA

Rest of Pol III
   distributive to
  processive
  (5x105 nts)
   β: clamp
 β-subunit dimer tethers the core of
E. coli DNA polymerase III to DNA
    E. coli DNA polymerase III
  γ complex: (γ, δ, δ’, χ,
  and Ψ)
   – Loading of the b-
     subunit clamp
      • ATP
   – Unloading
• τ subunit
   – Dimerize two core
     polymerase
Chain Elongation
Questions?
              Growing Fork
• Leading
  strand:
  continuous
• Lagging
  strand:
  discontinuous
        Lagging strand synthesis
• Leading strand:
  continuous
• Lagging strand:
  discontinuous
• Okazaki fragment:
  – Reiji Okazaki
  – Bac: 1-2 kb
  – Euk: 100-200 bp
Gap Removal
   • Pol I
      – 5’-3’
        exonuclease
      – Fills gap


   • Ligase
Ligase
Okazaki's Experiments on the Mechanism of DNA
          Replication in Bacterial Cells
    Fidelity of DNA Replication
  α subunit of Pol III error rate: 1 in 104
• Observed mutation rate: 10-9

• 3’-5’ proofreading exonuclease activity of DNA
  polymerase
   – E. coli Pol I
   – E. coli Pol III ε subunit.
• Mismatch repair system that distinguish newly
  synthesized DNA from the old one.

• Experiments?
Proofreading by 3’ to 5’
     Exonuclease
   Proofreading by 3’ to 5’
Exonuclease activity of DNA Pol
DNA Pol I Structure
 A Summary of
DNA Replication
  in Bacteria
 A Summary of
DNA Replication
  in Bacteria
     DNA Replication Movie
• Replication-Lehninger.mov
     E. coli replication proteins at a
               growing fork
τ subunit
   Holds two core
   polymerase
   Contacts DnaB
   protein.
   Coupling of the Leading and
    Lagging Strand Synthesis

• Replication-leading lagging coupling.mov
  Properties of DNA Polymerase
• E. coli Polymerase
                       I   II   III
• Polymerization       +   +    +
• Exonuclease
  3’-5’                +   +    +
  5’-3’                +   -    -
• Synthesis from
  Intact DNA           -   -    -
  Primed Single Strand +   -    -
  Primed Single Strand +   -    +
       +SSB
Overwinding that occurs when
 duplicating circular DNA.
              Topoisomerase I




• Nicking and then closing one strand of dsDNA
            Topoisomerase II

• Breaking and
  rejoining double-
  strand DNA
   Topoisomerase II
separates chromosomes
  at the end of DNA
      replication.
  Resolve the Supercoils During
        DNA Replication

• Replication-twist of DNA.mov
Eukaryotic
Replication
Machinery
Eukaryotic
Replication
Machinery
Loading
of PCNA
PCNA and β Clamp
 Properties of DNA Polymerase
• Mammalian Polymerase
   α
   β
   γ
   δ
   ε
Replication of Circular DNA
 The End-
Replication
 Problem
    The
Extension of
Telomeres by
 Telomerase
   Movie: DNA Replication at
      Chromosome End

• Replication-chromosome end.mov
 Summary of DNA Replication
• General Features
   – Semiconservative
   – Bidirectional
   – Replication origin
• DNA replication machinery
   –   Initiation proteins
   –   Helicase
   –   Primase
   –   Polymerases
   –   Leading and lagging strands
   –   Telomerase
• Topoisomerases in DNA replication
Questions?
                 Reference
•   Biochemistry, Stryer
•   Biochemistry, Lehninger
•   The World of the Cell, Becker et al
•   Molecular Cell Biology, Lodish et al

				
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posted:8/4/2010
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Description: Genetics of Respiratory Disease