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Structural Origin of Fidelity in DNA directed DNA polymerases BIOC 568 Sept. 2, 2009

Structural Origin of Fidelity in DNA directed DNA polymerases BIOC 568 Sept. 2, 2009. Polymerase active site. Active site metal ion. DNA pol I – right hand analogy. fingers. palm. thumb. 3’->5’ exonuclease. Fingers in blue Thumb in green Palm in red. Template Primer.

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Structural Origin of Fidelity in DNA directed DNA polymerases BIOC 568 Sept. 2, 2009

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  1. Structural Origin of Fidelity in DNA directed DNA polymerases BIOC 568 Sept. 2, 2009

  2. Polymerase active site Active site metal ion DNA pol I – right hand analogy fingers palm thumb 3’->5’ exonuclease

  3. Fingers in blue Thumb in green Palm in red

  4. Template Primer Template follows ‘wall’ Of fingers, doesn’t go through Thumb and fingers Polymerase Active site Think about directionality of strands: 5’->3’ 1LV5

  5. Template Primer 3’->5’ exonuclease Active site Think about directionality of strands: 5’->3’ 1KLN

  6. 5’->3’ exonuclease 1CMW taq DNA polymerase

  7. Structural origin of fidelity – 3KTQ, ternary complex with taq pol. Fingers close around incoming dNTP/template bp

  8. 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

  9. 2+ 2+ Mg Mg 2 metal ion mechanism O O C O - O O 3’ O P P - O O O - O P O - - O COSB 8,54

  10. Structural origin of fidelity – closure of fingers can only happen with correct bp. Mispairs would collide with surrounding protein Taq DNA pol 3KTQ

  11. Structural origin of fidelity – closure of fingers can only happen with correct bp. Mispairs would collide with surrounding protein Taq DNA pol 3KTQ

  12. RB69 DNA polymerase

  13. Structural origin of fidelity – if rxn happens with open conformation, the lack of minor groove interactions weakens complex favoring dissociation to 3’->5’ exo site

  14. 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

  15. After Translocation-4KTQ binary complex of taq pol with primer/template Tyr 671 dNTP binding site Next template base Sharp kink in template

  16. 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

  17. After Translocation-4KTQ binary complex of taq pol with primer/template Tyr 671 Phe 667 Next template base dNTP Sharp kink in template

  18. Dpo4 Translation polymerase from the Archaea Sulfolobus solfataricus Much more open pocket Incoming nucleotide (red) and template base (black)

  19. Dpo4 translesion DNA polymerase with benzopyrene adduct on G Notice the syn angles Adducted G adduct Skipped the adducted nucleotide by flipping it away

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