C. del Barrio, L. Embún, B. García, G. Miró, R. Pinacho, D. S. Rivera

1. DNA POLYMERASES: A Structural Approach C. del Barrio, L. Embún, B. García, G. Miró, R. Pinacho, D. S. Rivera

2. Table of contents • INTRODUCTION • STRUCTURAL DESCRIPTION • DNA SYNTHESIS PATHWAY • DNA-ENZYME INTERACTIONS • ACTIVE SITE • EVOLUTIVE FEATURES • CONCLUSIONS

3. DNA Replication between cell divisions Prokaryotes during S phase preceding mitosis or meiosis I Eukaryotes • Process of copying a double-stranded DNA molecule • Important in all known life forms • Each DNA strand holds the same information, so both strands can serve as templates for the reproduction of the opposite strand • Template strand is preserved and new strand is assembled from nucleotides: semiconservative replication • Resulting double-stranded DNA molecules are identical • Proofreading and error-checking mechanisms ensure fidelity • DNA replication is also performed in the laboratory: PCR

4. DNA Replication adds nucleotides in 5’-3’ until it encounters RNA primer of previous Okazaki fragment 3’ Daughter duplex 5’ removes the ribonucleotide primer (5’-3’ nuclease activity) Leading strand Parental DNA duplex 5’ Short RNA primer 3’ DNA pol I Okazaki fragment continues DNA synthesis by filling the gaps with deoxyribonucleotides (polymerase activity) Direction of fork movement Lagging strand Point of joining 5’ • Synthesis of DNA proceeds from a replication fork where the strands of the parent DNA are separated • Both strands serve as templates for replication during which new DNA strands are 5’-3’ synthesized • One strand is synthesized as a continuous chain • The other strand (which uses 5’-3’ parent strand as template) is made as a series of short DNA molecules: Okazaki fragments • Okazaki fragments require an RNA primer at 5’ ends to initiate DNA synthesis started by DNA pol III

5. DNA Replication  OTHER IMPORTANT ENZYMES INVOLVED IN DNA REPLICATION • Helicase • Separates the double-helical configuration • Topoisomerase • Catalyzes and guides unknotting of DNA (topological unlinking of the 2 strands) • SSB / SSBP • Bind to single-strands, keeping them separated and allowing DNA replication machinery to perform its function • RNA primase • Performs new RNA primer synthesis • No known DNA pol can initiate the synthesis of a DNA strand without initial RNA primers • DNA ligase • Its nick sealing joins new Okazaki fragment to the growing chain

6. DNA polymerase families

7. DNA polymerase I lineage

8. DNA polymerase I domains polymerase 3’-5’ exonuclease 5’-3’ exonuclease DNA template 3’ 5’ 5’ 3’ primer N C 518 residue number 928 324 1 Klenow fragment distance between polymerase active site and exonuclease binding site = 30 Å

9. DNA polymerase I functions 3’ -OH 5’ C T T T T primer strand A A A A A A A A A 5’ -OH 3’ 5’ 3’ T T T T T A A A A A A A A A 3’ 5’ template strand this strand determines which deoxyribonucleotide will be added POLYMERASE REACTION • It catalyzes stepwise addition of a deoxyribonucleotide to 3’-OH end of the primer strand that is paired to a second template strand • The new strand grows in 5’-3’ direction • Each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by pol

10. DNA polymerase I functions 3’ 5’ A A A A A A A A A A A T T T T T T T T T G 3’ 5’ 3’ hydrolysis site 5’-3’ EXONUCLEASE ACTIVITY • The second domain hydrolyzes DNA starting from the 5’ end of DNA • It can occur at the 5’ terminus or at a bond several residues away from it • The cleaved bond must be in a double-helical region 3’ 5’ A A A A A A A A A A A T T T T T T T T T G 3’ 5’ 5’ hydrolysis site 3’-5’ EXONUCLEASE ACTIVITY • One domain catalyzes hydrolysis of nucleotides at the 3’ end of DNA chains • To be removed, a nucleotide must have a free 3’-OH terminus and must not be part of a double helix

11. Taq DNA polymerase I: structure and domains

12. Why do we use Taq polymerase I instead of the one from E. coli? RMS: 1.75 Taq polymerase I (Klentaq fragment) [3KTQ] E. coli polymerase I(Klenow fragment) [1D8YA]

13. Taq polymerase I N-term C-term PDB: 1CMW

14. Taq polymerase 5’-3’ exonuclease domain Klentaq fragment Linker region (links Klentaq to N-term fragment) PDB: 1CMW

15. 5’-3’ exonuclease domain N-term resolvase-like domain C-term SAM fold PDB: 1CMW

16. Klentaq fragment Fingers Thumb Palm 3’-5’ exonuclease-like domain Linker region (links to N-term fragment) PDB: 3KTQ

17. Klentaq fragment Fingers Thumb Palm 3’-5’ exonuclease-like domain PDB: 3KTQ

18. DNA polymerase I Klenow fragment N C 8 14 9 13 12 7 LARGE DOMAIN • α+β Type with 6-stranded antiparallel β sheet • Connection between β strands 9 and 12 makes a long excursion that builds up one side of the DNA-binding cleft • It contains helices L-Q as well as the antiparallel hairpin of β strands 11 and 12

19. DNA polymerase I Klentaq fragment LARGE DOMAIN PDB: 3KTQ

20. DNA pol Klenow fragment nucleotide binding site C 5 2 3 4 1 SMALL DOMAIN • α/β Type with 1 antiparallel βstrand • 5-stranded βsheet with 2 connecting helices and 1 helix (C) at the carboxy terminus (E.coli) UNIQUE TOPOLOGY FOR A MIXED β SHEET

21. Highly conserved regions

22. Highly conserved regions Region 1 Region 2 Motif A Motif B Region 6 Motif C

23. Asp610 Hydrophobic residues Motif A “DYSQIELR”

24. Arg659 Lys663 Gly668 Tyr671 Motif B “RRxhKhhNFGhhY”

25. His784 “HDE” Asp785 Motif C

26. PATHWAY OF DNA SYNTESIS Part 1: DNA binding

27. Pathway of DNA Synthesis PPi dNTP E+TP E-TP E-TP-dNTP E-TPn+1-PPi E-TPn+1 4 1 2 3 1 Polymerase (E) binds with template-primer (TP) 2 Appropriate dNTP binds with polymerase-DNA complex 3 Nucleophilic attack results in phosphodiester bond formation 4 Pyrophosphate (PPi) is released phosphodiester bond formation rate limiting step conformational change preceding nucleotide incorporation dynamic interactions between polymerase with its nucleic acid and dNTP substrates

28. Pathway of DNA Synthesis PPi dNTP E+TP E-TP E-TP-dNTP E-TPn+1-PPi E-TPn+1 4 1 2 3 • Polymerases undergo 4 significant conformational changes: • During DNA binding step • Subsequent to dNTP binding step and immediately preceding chemical catalysis • Subsequent to nucleotide incorporation during PPi release • During translocation towards new primer 3’-OH terminus

29. Template Region 1 First step: polymerase binds to template PDB: 2KTQ

30. REGION 1 2.40 Å 3.21 Å DNA PDB: 2KTQ Open conformation 1

31. Low dNTPs concentration OPEN CONFORMATION Stacking interaction 2 Tyr671 DCT 3 1st base 2.40Å 90º 1st base PDB: 2KTQ

32. DCT PDB: 2KTQ

33. When dNTPs concentration increases... CLOSED CONFORMATION RMS: 0.75 Open conformation (2KTQ) Closed conformation (3KTQ)

34. Open conformation (2ktq.pdb) Closed conformation (3ktq.pdb) 40º 11-12 Å O-helix

35. O-helix Arg587 Arg659 His639 Lys663 Gln613 Tyr671 Asp785 Asp610 Closed conformation PDB: 3KTQ

36. Hydrophobic pocket 4.11Å Phe667 Tyr671 Tyr671 PDB: 3KTQ

37. Hydrogen bonding dNTP ~3Å Hydrogen bonds PDB: 1QSS

38. Structure dNTP Pentose • Base Triphosphate α γ β PDB: 1QSS

39. Electrostatic Interaction α- phosphate Arg587 - 5,6Å + PDB: 1QSS

40. Electrostatic Interaction Gln613 + 4,07Å - β phosphate PDB: 1QSS

41. Electrostatic Interaction Lys663 + - 5,02Å PDB: 1QSS β phosphate Β-

42. Electrostatic Interaction His639 + - γ phosphate ~5Å PDB: 1QSS

43. Electrostatic Interaction + - Arg659 2,45 Å γ phosphate PDB: 1QSS

44. Stacking interactions Phe667 Tyr671 3,19Å 4,23Å Base PDB: 1QSS

45. Metal-mediated interactions triphosphate Mg Asp785 Catalytic site Asp610 PDB: 3KTQ

46. Taq polymerase: Active site Catalytic Triad: 3 active site carboxylates Motif A Asp610 Motif C Asp 785 Glu 786 GLU 786 ASP 785 Equivalence in Escherichia coli: Asp 705 Glu 710 Asp 882 Glu 883 ASP 610 2 viable triads!! PDB: 3KTQ

47. Taq polymerase: Active site Mg A Catalytic Triad: Mg B PDB: 3KTQ

48. Taq polymerase: Active site Coordination of the Mg B Pα Pβ Pγ Tyr 611 Asp 785 Asp 610 PDB: 3KTQ

49. Taq pol : Active site PO4β PO4γ Asp 785 Asp 610 Tyr 611 Coordination of the Mg B PO4α PDB: 3KTQ

50. Taq pol : Active site Coordination of the Mg A PO4 α Asp 785 O- MgA OH2O O- 3’OH- O- OH2O Asp 610 PDB: 3KTQ