Isolation of DNA polymerase I

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Isolation of DNA polymerase I

• A. Kornberg (1956) isolated a DNA polymerizing
  activity from E. coli.
• Required a template
• Required a primer
• Synthesized the complement of the template
• Size 928 amino acids, 103 kDa
• Encoded by polA gene

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Reaction catalyzed by DNA Pol I

• Add dNMP (from dNTP) to 3 end of a growing chain
• Release pyrophosphate (PPi)
• Reversible pyrophosphorolysis
• Requires template, primer, Mg, and all 4 dNTPs

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DNA Pol I adds a nucleotide and releases PPi
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Subsequent hydrolysis of pyrophosphate
Catalyzed by a separate enzyme
pyrophosphatase Helps drive the reaction forward
(toward synthesis)
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Exo- vs. Endonucleases

• Exonucleases remove nucleotides from the ends of
  DNA (and/or RNA) molecules.
• Catalyze hydrolysis of phosphodiester bonds
• Will NOT work on circular DNA
• Endonucleases cleave in the middle of DNA (and/or
  RNA) molecules.
• Catalyze hydrolysis of phosphodiester bonds
• DO work on circular DNA

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DNA Pol I is a 3 to 5 exonuclease

• Provides a proofreading function
• If an incorrect nucleotide is added, the enzyme
  recognizes the mismatch and removes the incorrect
  nucleotide.
• The incorrect nucleotide is removed by
  hydrolysis.
• Proofreading is common to many but not all DNA
  polymerases.

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Chain elongation by DNA polymerase I
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Excision of incorrect nucleotide by 3-5
exonuclease activity of DNA polymerase I
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DNA Pol I is a 5 to 3 exonuclease

• Removes nucleotides in base-paired regions
• Can remove DNA or RNA
• Physiological function appears to be repair of
  damaged DNA and removal of RNA primers from
  Okazaki fragments
• Can be used to label DNA in vitro by nick
  translation
• Not a common activity of other DNA polymerases

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5 to 3 exonuclease activity
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DNA Pol I is a multi-domain protein

• Cleavage by the protease subtilisin can separate
  the 5 to 3 exo from the polymerase and
  proofreading 3 to 5 exo.
• Polymerase 3 to 5 exonuclease are in the
  remaining Klenow fragment
• Polymerase active site is in the palm of the
  cupped right hand
• 3 to 5 exonuclease is about 25 Angstroms away
• Klenow fragment has two functional domains

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3 functional domains in 1 polypeptide
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Polymerase domain resembles a cupped right hand
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Many DNA polymerases have a similar structure

• DNA polymerase from Thermus aquaticus
• Polymerase
• 3 to 5 exo domain is present but inactive
• 5 to 3 exo is also present
• Cupped right hand structure also seen in
• T7 RNA polymerase
• HIV reverse transcriptase (RNA dependent DNA
  polymerase
• Others
• DNA Pol I founding member of ONE class of
  polymerases

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Null mutants at polA (encoding DNA Pol I) are
viable!
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DNA polymerase III

• Enzyme used during replication
• Multisubunit protein
• High processivity

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DNA polymerase III DNA Pol III

• Discovered in extracts of polA- cells, i.e.
  lacking DNA Pol I
• DNA Pol III is the replicative polymerase
• Loss-of-function mutations in the genes encoding
  its subunits block DNA replication (dna mutants)
• Highly processive
• Multiple subunits
• Also discovered DNA Pol II in polA- extracts
  (role in DNA repair)

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DNA Pol III Low abundance but high processivity
Comparison Pol I Pol III core Pol III
holo molecules per cell 400 40 10 nts
polymerized min-1 (molecule enz)-1 600 9000 42,
000 processivity nts polymerized per
initiation 3-188 10 gt105 5' to 3'
polymerase 3' to 5' exo,
proofreading 5' to 3' exo - -
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Processivity

• Amount of polymerization catalyzed by an enzyme
  each time it binds to a template.
• Measured in nucleotides polymerized per
  initiation
• High processivity of DNA Pol III results from
  activities of non-polymerase subunits

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Subunits of DNA Pol III and functions
Functional Mass component Subunit
(kDa) Gene Activity Core polymerase a 129.9 polC
dnaE 5' to 3' polymerase e 27.5 dnaQmutD 3'-
5' exonuclease q 8.6 Stimulates e
exonuclease Linker protein t 71.1 dnaX Dimerize
s cores Clamp loader g 47.5 dnaX Binds ATP
(aka g complex) d 38.7 Binds to b
(ATPase) d' 36.9 Binds to g and
b c 16.6 Binds to SSB y 15.2
Binds to c and g Sliding
clamp b 40.6 dnaN Processivity factor
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Synergism among subunits

• Activities of polymerase (a) and 3 to 5
  exonuclease (e) are higher in the core (aeq) than
  in individual subunits.
• The g complex has 6 subunits that work together
  to load and unload the clamp (b2) for processive
  synthesis

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Subassemblies have distinct functions
e
e
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Processivity factor beta2 Sliding clamp

• The b subunit forms a homodimer.
• The structure of this homodimer is a ring.
• The ring encloses DNA, thereby clamping the DNA
  Pol III holoenzyme to the template.
• An enzyme that is clamped on cannot come off
  easily, and thus will be highly processive.

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Gamma complex Clamp loader/unloader

• The g complex (g2dd'cy) loads the b dimer clamp
  onto a primer-template.
• Bind the clamp (b dimer) onto the loader (g
  complex) need ATP
• Exchange the clamp from the loader to the core
  need ATP hydrolysis
• Unload the clamp when polymerase reaches a
  previously synthesized Okazaki fragment need ATP
  
• The ATP-bound form of the g complex can bind the
  clamp
• The ADP-bound form releases the clamp

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Loading the beta2 clamp
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Unloading the beta2 clamp
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Model for gamma complex loading beta clamp
Jeruzalmi, ODonnell and Kuriyan (2001) Cell 106
429-441
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Asymmetric dimer of DNA PolIII simultaneous
replication of both strands of DNA

• The 2 catalytic cores of DNA Pol III are joined
  by the tau subunits to make an asymmetric dimer.
• Model one holoenzyme synthesizes both strands at
  a replication fork.
• One core synthesizes the leading strand
• Other synthesizes the lagging strand.
• If the template for lagging strand synthesis is
  looped around the enzyme, then both strands are
  synthesized in the direction of fork movement.

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One holoenzyme, 2 templates
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Simultaneous replication of both strands of DNA
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Eukaryotic replicative DNA polymerases

• Nuclear DNA replication
• a primase plus low processivity polymerase
• d both leading and lagging strand synthesis
• e may be used in lagging strand synthesis

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Eukaryotic DNA polymerases in replication
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PCNA is homologous to beta2 clamp
Mammalian PCNA is a trimer, each monomer of
which has two similar domains.
The beta subunit of E. coli DNA Pol III is a
dimer, each monomer of which has three similar
domains.
The domains in each are very similar, and the net
result is a ring of 6 domains. The two proteins
are structurally homologous and have similar
functions.
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Similarities between bacterial and eukaryotic
replication machinery
Function E. coli Pol III Eukaryotic Leading and
lagging asymmetric polymerase d strand
synthesis dimer Sliding clamp b subunit
PCNA Clamp loader g-complex RFC Primase DnaG
polymerase a Single strand binding SSB RFA Sw
ivel Gyrase (Topo II) Topo I or II (Maintain
DNA topology)
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Additional eukaryotic DNA polymerases

• Nuclear repair DNA polymerases b and e
• Mitochondrial DNA replication g
• Plus
• Reverse transcriptase
• Terminal deoxynucleotidyl transferase
• Telomerase

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