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REPLICATION

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					REPLICATION
   Chapter 7
               The Problem

   DNA is maintained in a compressed,
    supercoiled state.
   BUT, basis of replication is the formation
    of strands based on specific bases pairing
    with their complementary bases.
    Before DNA can be replicated it must be
    made accessible, i.e., it must be unwound
Models of Replication




THREE HYPOTHESES FOR DNA REPLICATION
     MODELS OF DNA REPLICATION
(a) Hypothesis 1:    (b) Hypothesis 2:         (c) Hypothesis 3:
Semi-conservative   Conservative replication   Dispersive replication
   replication




                     Intermediate molecule
PREDICTED
DENSITIES OF
NEWLY
REPLICATED
DNA
MOLECULES
ACCORDING
TO THE
THREE
HYPOTHESES
ABOUT DNA
REPLICATION
        Meselson and Stahl
Conclusion: Semi-conservative replication of DNA
         Replication as a process
   Double-stranded DNA unwinds.

The junction of the unwound
molecules is a replication fork.

A new strand is formed by pairing
complementary bases with the
old strand.

Two molecules are made.
Each has one new and one old
DNA strand.
          Extending the Chain
   dNTPs are added individually
   Sequence determined by pairing with
    template strand
   DNA has only one phosphate between
    bases, so why use dNTPs?
Extending the Chain
                DNA Synthesis
                3’-OH nucleophilic attack
                on alpha phosphate of
                incoming dNTP




               removal and splitting of pyrophosphate
               by inorganic pyrophosphatase

2 phosphates
Chain Elongation in the 5’  3’ direction
    Semi-discontinuous Replication

   All known DNA pols work in a 5’>>3’
    direction
   Solution?
       Okazaki fragments
Okazaki Experiment
     Continuous synthesis




     Discontinuous synthesis

DNA replication is semi-discontinuous
     Features of DNA Replication
   DNA replication is semiconservative
       Each strand of template DNA is being copied.
   DNA replication is semidiscontinuous
       The leading strand copies continuously
       The lagging strand copies in segments (Okazaki
        fragments) which must be joined
   DNA replication is bidirectional
       Bidirectional replication involves two replication
        forks, which move in opposite directions
    DNA Replication-Prokaryotes
   DNA replication is semiconservative.
    the helix must be unwound.
   Most naturally occurring DNA is slightly
    negatively supercoiled.
 Torsional     strain must be released
   Replication induces positive supercoiling
 Torsional    strain must be released,
    again.
   SOLUTION: Topoisomerases
The Problem of Overwinding
        Topoisomerase Type I

 Precedes replicating DNA
 Mechanism

     Makes a cut in one strand, passes other
      strand through it. Seals gap.
     Result: induces positive supercoiling as
      strands are separated, allowing
      replication machinery to proceed.
                    Helicase
 Operates in replication
  fork
 Separates strands to
  allow DNA Pol to
  function on single
  strands.
Translocate along single
  strain in 5’->3’ or 3’->
  5’ direction by
  hydrolyzing ATP
    Gyrase--A Type II Topoisomerase
   Introduces negative supercoils
   Cuts both strands
   Section located away from actual cut is
    then passed through cut site.
          Initiation of Replication

   Replication initiated at specific sites:
    Origin of Replication (ori)
   Two Types of initiation:
       De novo –Synthesis initiated with RNA
        primers. Most common.
       Covalent extension—synthesis of new strand
        as an extension of an old strand (―Rolling
        Circle‖)
De novo Initiation
                  Binding to Ori
                   C by DnaA
                   protein
                  Opens
                   Strands
                  Replication
                   proceeds
                   bidirectionally
    Unwinding the DNA by Helicase
           (DnaB protein)
   Uses ATP to separate the DNA strands
   At least 4 helicases have been identified in
    E. coli.
   How was DnaB identified as the helicase
    necessary for replication?
   NOTE: Mutation in such an essential gene
    would be lethal.
   Solution?
       Conditional mutants
Liebowitz Experiment


            What would you
            expect if the
            substrates are
            separated by
            electrophoresis after
            treatment with a
            helicase?
Liebowitz Assay--Results
                  What do these
                   results indicate?
                  ALTHOUGH PRIMASE
                   (DnaG) AND SINGLE-
                   STRAND BINDING
                   PROTEIN (SSB) BOTH
                   STIMULATE DNA
                   HELICASE (DnaB),
                   NEITHER HAVE
                   HELICASE ACTIVITY
                   OF THEIR OWN
      Single Stranded DNA Binding
             Proteins (SSB)
   Maintain strand separation once helicase
    separates strands
   Not only separate and protect ssDNA, also
    stimulates binding by DNA pol (too much
    SSB inhibits DNA synthesis)
   Strand growth proceeds 5’>>3’
        Replication: The Overview
   Requirements:
       Deoxyribonucleotides
       DNA template
       DNA Polymerase
          5 DNA pols in E. coli
          5 DNA pols in mammals

       Primer
   Proofreading
     The DNA Polymerase Family
 A total of 5 different DNAPs have been reported
                        in E. coli
 DNAP I: functions in repair and replication
 DNAP II: functions in DNA repair (proven in 1999)

 DNAP III: principal DNA replication enzyme

 DNAP IV: functions in DNA repair (discovered in 1999)

  DNAP V: functions in DNA repair (discovered in 1999)



    To date, a total of 14 different DNA polymerases
            have been reported in eukaryotes
                  DNA pol I
   First DNA pol discovered.
   Proteolysis yields 2 chains
      Larger Chain (Klenow Fragment) 68 kd
         C-terminal 2/3rd. 5’>>3’ polymerizing
          activity
         N-terminal 1/3rd. 3’>>5’ exonuclease
          activity
      Smaller chain: 5’>>3 exonucleolytic
       activity
         nt removal 5’>>3’
         Can remove >1 nt
         Can remove deoxyribos or ribos
                  DNA pol I
   First DNA pol discovered.
   Proteolysis yields 2 chains
      Larger Chain (Klenow Fragment) 68 kd
         C-terminal 2/3rd. 5’>>3’ polymerizing
          activity
         N-terminal 1/3rd. 3’>>5’ exonuclease
          activity
      Smaller chain: 5’>>3 exonucleolytic
       activity
         nt removal 5’>>3’
         Can remove >1 nt
         Can remove deoxyribos or ribos
The structure of the
Klenow fragment of
DNAP I from E. coli
     Nick Translation
Requires 5’-3’ activity of DNA
  pol I
Steps
1. At a nick (free 3’ OH) in the DNA the
   DNA pol I binds and digests
   nucleotides in a 5’-3’ direction
2. The DNA polymerase activity
   synthesizes a new DNA strand
3. A nick remains as the DNA pol I
   dissociates from the ds DNA.
4. The nick is closed via DNA ligase




                                           Source: Lehninger pg. 940
              Nick Translation 2

    5'-exonuclease activity, working together with
    the polymerase, accomplishes "nick translation"




     This activity is critical in primer removal
DNA Polymerase I is great, but….
    In 1969 John Cairns and Paula deLucia
-isolated a mutant bacterial strain with only 1%
   DNAP I activity (polA)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could
  divide, so obviously it can replicate its
  DNA
Conclusion:
 DNAP I is NOT the principal replication
  enzyme in E. coli
            Other clues….
- DNAP I is too slow (600 dNTPs added/minute)
- DNAP I is only moderately processive
  (processivity refers to the number of dNTPs
  added to a growing DNA chain before the
  enzyme dissociates from the template)

Conclusion:
 There must be additional DNA polymerases.

 Biochemists purified them from the polA
  mutant
            DNA Polymerase III
    The major replicative polymerase in E. coli
   ~ 1,000 dNTPs added/sec
   It’s highly processive: >500,000 dNTPs
    added before dissociating
   Accuracy:
      1 error in 107 dNTPs added,

      with proofreading final error rate of 1 in
       1010 overall.
DNA Polymerase III Holoenzyme (Replicase)
      The 10 subunits of E. coli DNA polymerase III
                    Subunit Function

                        a    5’ to 3’ polymerizing activity
               Core
               enzyme
                        e    3’ to 5’ exonuclease activity
                        q    a and e assembly (scaffold)
                        t    Assembly of holoenzyme on DNA
  Holoenzyme




                        b    Sliding clamp = processivity factor
                        g    Clamp-loading complex
                        d    Clamp-loading complex
                        d’   Clamp-loading complex
                        c    Clamp-loading complex
                        y    Clamp-loading complex
    Activities of DNA Pol III
 ~900 kd
 Synthesizes both leading and lagging
  strand
 Can only extend from a primer (either
  RNA or DNA), not initiate
 5’>>3’ polymerizing activity

 3’>>5’ exonuclease activity

 NO 5’>>3’ exonuclease activity
     The 5’ to 3’ DNA polymerizing activity




Subsequent
hydrolysis of
PPi drives the
reaction forward




            Nucleotides are added at the 3'-end of the strand
    Leading and Lagging Strands
   REMEMBER: DNA polymerases require a
    primer.
   Most living things use an RNA primer
   Leading strand (continuous): primer made
    by RNA polymerase
   Lagging strand (discontinuous): Primer
    made by Primase
       Priming occurs near replication fork, need to
        unwind helix. SOLUTION: Helicase
       Primosome= Primase + Helicase
The Replisome
   DNA pol III extends on
    both the leading and
    lagging strand
   Growth stops when Pol
    III encounters an RNA
    primer (no 5’>>3’
    exonuclease activity)
   Pol I then extends the
    chain while removing
    the primer (5’>>3’)
   Stops when nick is
    sealed by ligase
            Ligase
   Uses NAD+ or ATP for
    coupled reaction
   3-step reaction:
       AMP is transferred to Lysine
        residue on enzyme
       AMP transferred to open 5’
        phosphate via temporary
        pyrophosphate
       AMP released, phosphodiester
        linkage made
   NADNMN + AMP
   ATP ADP + PPi
DNA Replication Model
       1.   Relaxation of supercoiled
            DNA.
       2.   Denaturation and untwisting
            of the double helix.
       3.   Stabilization of the ssDNA in
            the replication fork by SSBs.
       4.   Initiation of new DNA
            strands.
       5.   Elongation of the new DNA
            strands.
       6.   Joining of the Okazaki
            fragments on the lagging
            strand.
                                 Termination of
                                  Replication

                                  Tus Protein-arrests
                                  replication fork
                                  motion

   Occurs @ specific site opposite ori c
   ~350 kb
   Flanked by 6 nearly identical non-palindromic*,
    23 bp terminator (ter) sites
   * Significance?
     FIDELITY OF REPLICATION
 Expect 1/103-4, get 1/108-10.
 Factors
     3’5’ exonuclease activity in DNA pols
     Use of ―tagged‖ primers to initiate
      synthesis
     Battery of repair enzymes

     Cells maintain balanced levels of dNTPs
            Why Okazaki Frags?
   Or, why not 3’5’ synthesis?
   Possibly due to problems with proofreading.
   PROBLEM:
       Imagine a misincorporation with a 3’5’
        polymerase
       How is it removed?
       How is the chain extended?
       Is there a problem after removing a mismatch?

				
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