Structural Origin of Fidelity in

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Structural Origin of Fidelity in DNA directed
DNA polymerases BIOC 568 Sept. 2, 2009
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DNA pol I right hand analogy
fingers
palm
thumb
3-gt5 exonuclease
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Fingers in blue Thumb in green Palm in red
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Template Primer
Template follows wall Of fingers, doesnt go
through Thumb and fingers
Polymerase Active site
Think about directionality of strands 5-gt3
1LV5
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Template Primer
3-gt5 exonuclease Active site
Think about directionality of strands 5-gt3
1KLN
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5-gt3 exonuclease
1CMW taq DNA polymerase
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Structural origin of fidelity 3KTQ, ternary
complex with taq pol. Fingers close around
incoming dNTP/template bp
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Structural origin of fidelity 3KTQ, ternary
complex with taq pol. Fingers closed around dNTP
Bp to template base

• Basic residues from
• fingers when fingers closed
• accelerates reaction
• but only when correct bp
• (since fingers close only
• on correct bp)

Incoming dNTP
2 metal ion mechanism
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2 metal ion mechanism
O
O
C
O
-
O
3
O
P
O
P
-
O
O
O
-
O
P
O
-
-
O
COSB 8,54
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Structural origin of fidelity closure of
fingers can only happen with correct bp.
Mispairs would collide with surrounding protein
Taq DNA pol 3KTQ
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Structural origin of fidelity closure of
fingers can only happen with correct bp.
Mispairs would collide with surrounding protein
Taq DNA pol 3KTQ
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RB69 DNA polymerase
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Structural origin of fidelity if rxn happens
with open conformation, the lack of minor groove
interactions weakens complex favoring
dissociation to 3-gt5 exo site
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Translocation Release of pyrophosphate breaks
the electrostatic interactions that help to hold
the fingers down. Fingers open, the
primer-template shifts 1bp, with the tyrosine
occupying the position of the next template base.

• Basic residues from
• Fingers when fingers closed
• Accelerates reaction
• But only when correct bp
• (since fingers close only
• on correct bp)

Incoming dNTP
2 metal ion mechanism
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After Translocation-4KTQ binary complex of taq
pol with primer/template
Tyr 671
dNTP binding site
Next template base
Sharp kink in template
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After Translocation-4KTQ binary complex of taq
pol with primer/template
Tyr 671
Only 1 metal ion
dNTP
Next template base
Sharp kink in template
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After Translocation-4KTQ binary complex of taq
pol with primer/template
Tyr 671
Phe 667
Next template base
dNTP
Sharp kink in template
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Dpo4 Translation polymerase from the Archaea
Sulfolobus solfataricus
Much more open pocket
Incoming nucleotide (red) and template base
(black)
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Dpo4 translesion DNA polymerase with benzopyrene
adduct on G
Notice the syn angles
Adducted G
adduct
Skipped the adducted nucleotide by flipping it
away