Chapter 10 � DNA Replication

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Chapter 10 � DNA Replication Powered By Docstoc
					Chapter 12 – DNA Replication
    Replication models for DNA
• Conservative
   – DNA molecule remains intact and entire molecule serves as a
     template
   – Results in one completely old molecule, and one completely new
     molecule
• Dispersive
   – DNA molecule breaks into fragments, which serve as template
     and then reassemble
   – Each molecule a mixture of old and new
• Semi-conservative
   – DNA separates into two strands, and each serves as a template
   – Each molecule consists of one old strand and one new strand
Meselson and Stahl experiment
• Equilibrium density
  gradient centrifugation
   – Heavy salt solution used to
     measure substances’
     densities
   – Denser molecules are
     lower in tube
• Radioactively labeled
  DNA with 15N – heavier
  than DNA with 14N
        Meselson and Stahl cont
• Grew E. coli in 15N, then
  swtiched to media with 14N

• First replication – intermediate
  band
    – Rules out conservative
      method

• Second replication – one 14N
  band (gets progressively
  darker with each division) and
  one intermediate band (gets
  progressively lighter with each
  division)
• Semi-conservative method
          Modes of Replication
• Replication origin
   – Starting point of replication
      • A-T rich regions
   – Bacterial chromosomes have one; eukaryotic
     chromosomes have many
   – Replicon – individual unit of replication
• 3 types of replication
   – Theta replication
   – Rolling circle replication
   – Linear replication
             Theta Replication
• Circular DNA in bacteria
• Replication bubble
  formed from DNA
  unwinding and strands
  separating
• Replication fork – point
  where two strands
  separate
• Continues bi-directionally
  until they meet
      Rolling Circle Replication
• Viruses and certain
  plasmids (F factor)
• One strand breaks, new
  nucleotides are added to
  3′ end using intact strand
  as template
   – New strand displaces old
     strand; old strand can
     become double-stranded
     based on complementarity
• Only one strand serves
  as a template
           Linear Replication
• Eukaryotic
  chromosomes
  – Each has many origins
    of replication
  – Each replicon is
    smaller than
    prokaryotic
    chromosome
• Replication forms
  eventually meet and
  replicons fuse
   Requirements for replication
• Single-stranded
  template DNA

• dNTPs
  – Deoxyribonucleoside
    triphosphates (2
    phosphates are
    removed)


• Enzymes and other
  proteins
                 DNA polymerase
• Can only add new nucleotides
  to the 3′ end
   – Replication in 5′→3′ direction
• Old 3′→5′ template strand can
  be replicated continuously
   – Leading strand
• Old 5′→3′ template strand is
  replicated in small fragments –
  Okazaki fragments
   – Lagging strand
   – As DNA unwinds, another
     fragment is produced
   – Rolling-circle method does
     NOT have lagging strand
       Bacterial DNA Replication
• 4 general stages
   –   Initiation
   –   Unwinding
   –   Elongation
   –   Termination


• Initiation
   – Initiator proteins bind to Ori
     and unwind small segment
        • Allows other molecules to
          bind to DNA
                            Unwinding
• DNA helicases
   – Break hydrogen bonds
     between 2 strands
   – Move in 5′→3′ direction
• Single-strand binding proteins
   – Prevents reannealing
• DNA gyrase
   – In front of replication fork
   – Unwinding causes
     supercoiling
   – Is a topoisomerase – makes
     double-stranded break, and
     then reseals break behind it
       • Releases tension
                 Unwinding cont
• Primers
  – DNA polymerase can’t
    initiate a new strand – it
    can only elongate an
    existing strand
  – Primase
      • RNA polymerase
      • Does not require a primer
      • Adds short stretch of RNA
        nucleotides which is later
        replaced by DNA
        nucleotides and ligated
        together
  – Leading strand requires
    one primer; lagging
    requires many
                    Elongation
• DNA polymerase III
  – Adds nucleotides to 3′ end
  – Has 3′→ 5′ exonuclease activity
     • Can backtrack and replace an incorrect nucleotide
  – Has high processivity
     • Stays attached to template for long time
• DNA polymerase I
  – Has same direction abilities as III; in addition has
    5′→3′ exonuclease activity
     • Removes RNA primer and replaces nucleotides with DNA
       nucleotides
                Elongation cont
• Phosphodiester bonds
  – Covalent bond formed
    between 5′ phosphate
    group of new nucleotide to
    3′ -OH group of last
    nucleotide
• DNA ligase
  – DNA poly I replaces primer
    – leaves nick between last
    replaced nucleotide and 1st
    original DNA nucleotide
  – Ligase creates
    phosphodiester bond to
    form continuous strand
              Termination
• When 2 replication forks meet or specific
  DNA sequence is encountered
  – Termination protein binds to sequence and
    blocks helicase binding
    Fidelity of DNA replication
• Complementary base pairing
• Proofreading
  – Incorrect alignment causes DNA poly III to backtrack
    and remove incorrect base
• Mismatch repair
  – Causes deformity in double strand
  – Old strand is methylated; new strand is not
     • Distinguishes strands
  – Incorrect nucleotide is excised out and replaced
       Eukaryotic replication
• Have multiple polymerases (greek letters)
  – alpha and delta are major ones
• Nucleosome
  – DNA coiled around 8 histone proteins
  – Newly synthesized DNA molecules are
    quickly re-associated with histones (a mix of
    old and newly made)
           Linear chromosomes
• Circular DNA has a free –
  OH group in front of
  primer for new nucleotide
  to attach to
• Linear chromosomes
   – After primer is removed at
     the end of the
     chromosome, there is no
     free –OH group
   – Chromosome would
     shorten with each
     replication, removing
     telomeres and destabilize
     chromosome
                     Telomerase
• Telomeres are short
  repeating sequences
• Telomerase is a
  ribonucleoprotein
   – RNA portion – 12-22
     complementary nucleotides
   – Protein portion – acts as an
     enzyme to extend 3′ end
     with complementary DNA
   – 2nd strand replication –
     unknown mechanism
Possible 2nd strand replication
           Telomerase cont
• Activity decreases/stops in most mature
  cells
  – May lead to cellular aging due to de-
    stabilization of chromosomes
  – Normal cells have a limited number of
    replications
  – Telomerase activity has been shown to
    continue in cancer cells – immortal cells

				
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posted:8/31/2012
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