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

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

3. Fingers in blue Thumb in green Palm in red

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

5. 3’->5’ exonuclease Active site TemplatePrimer 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 2 metal ion mechanism Basic residues from fingers when fingers closed -accelerates reaction but only when correct bp (since fingers close only on correct bp) Incoming dNTP Bp to template base

9. Mg 2+ Mg 2+ P O O O P O O O P O O O O C 3’O O - - - -- 2 metal ion mechanism 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. Taq DNA pol 3KTQ Structural origin of fidelity – closure of fingers can only happen with correct bp. Mispairs would collide with surrounding protein

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. 2 metal ion mechanism Basic residues from Fingers when fingers closed -Accelerates reaction But only when correct bp (since fingers close only on correct bp) Incoming dNTP

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

16. After Translocation-4KTQ binary complex of taq pol with primer/template dNTP Tyr 671 Sharp kink in template Next template base Only 1 metal ion

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

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

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