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DNA Polymerases

DNA Polymerases. Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain their integrity to ensure faithful transmission of genetic information from one generation to the next.

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DNA Polymerases

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  1. DNA Polymerases • Nucleotide polymerizing enzymes that are central players in DNA repair and replication, • the processes that duplicate genomes and maintain their integrity to ensure faithful transmission of genetic information from one generation to the next. Molecular biology of the cell / Bruce Alberts … [et al.].-- 4th ed.

  2. DNA Polymerase Families Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  3. DNA Polymerase Families Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  4. DNA Polymerase Families Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  5. DNA Polymerase Families Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  6. DNA Polymerase Families Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  7. Structures and Compositions Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  8. Structures and Compositions Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  9. Polymerization Molecular biology of the cell / Bruce Alberts … [et al.].-- 4th ed. Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  10. Two-metal Mechanism Science. 1994 Dec 23;266(5193):2022-5

  11. News & View • “We disagree with this conclusion and suggest an alternative interpretation of the structural data, namely, that there is no contradiction between the orientations of the DNA inferred from these structures; rather, the apparent inconsistency is the result of an inappropriate alignment of the pol 1 structure with the other polymerase structures.” T. A. Steitz • “Although the interpretations of the data from our structural studies of pol 13 contradict interpretations of polymerase data by other research groups, it does not follow that the proposal of Steitz et al. is the best solution to this conundrum.” H. Pelletier Science. 1994 Dec 23;266(5193):2025-6. Science. 1994 Dec 23;266(5193):2022-5

  12. Polymerases are Highly Diverse Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  13. Polymerases are Highly Diverse Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  14. Polymerases are Highly Diverse Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  15. Functions of DNA Polymerases

  16. Functions of DNA polymerases • Replicating Damaged DNA • DNA Repair • Nucleotide Exision Repair • Base Excision Repair • Interstrand Cross-Link Repair • Nonhomologous End-Joining of Double-Strand Breaks • Replicating Undamaged DNA • Sister Chromatid Cohesion • Mitochondrial DNA replication • Cell-Cycle Checkpoints • Replication Restart and Homologous Recombination • Mismatch Repair • Development of the Immune System

  17. DNA polymerases involved in DNA repair and replication Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  18. Functions of DNA polymerases • Replicating Damaged DNA • Translesion Synthesis • DNA Repair • Nucleotide Exision Repair Advances in Protein Chemistry, Vol. 69, 137-165, 2004

  19. Nat Cell Biol. 2006 Jun;8(6):547-9

  20. Translesion Synthesis Nat Cell Biol. 2006 Jun;8(6):547-9

  21. Biological consequences: Pol η • Xeroderma Pigmentosum Variant • caused by molecular alterations in the POLH gene (pol η) • major function of polη is to allow DNA translesion synthesis of UV-induced TT-dimers in an error-free manner; Exp Dermatol. 2003 Oct;12(5):529-36

  22. Y Family Structure Mol Cell. 2001 Aug;8(2):417-26           

  23. Nucleotide Excision Repair Nat Cell Biol. 2006 Jun;8(6):547-9.

  24. Nucleotide Excision Repair • Repair of Bulky Lesions • Recognition • Separation of the double helix (iii) Incision (iv) Excision (v) Synthesis (vi) Ligation • Types • Global Genome Repair • Transcription Coupled Repair Nat Cell Biol. 2006 Jun;8(6):547-9.

  25. How to Measure NER • Global Genome Repair • Can be assessed using the technique of unscheduled DNA synthesis (UDS) in which repair synthesis is measured quantitatively by the incorporation of 3H thymidine by cells that are not in the S phase of the cell cycle • Transcription-Coupled Repair • Can be assessed by recovery of RNA synthesis after UV irradiation

  26. Pol Kappa • Localization pattern J Cell Sci. 2005 Jan 1;118(Pt 1):129-36

  27. Pol Kappa • Disruption causes major UV sensitivity • Even though pol k can not on its own bypass either of the major UV lesions PNAS | November 26, 2002 | vol. 99 | no. 24 | 15548-15553

  28. The Y-family DNA polymerase kappa (pol kappa) functions in mammalian nucleotide-excision repair Ogi T, Lehmann AR. Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton,

  29. Complementation of the NER deficiency by polκ Nature Cell Biology8, 640 - 642 (2006)

  30. RNA Synthesis Recovery:TCR Nature Cell Biology8, 640 - 642 (2006)

  31. Unscheduled DNA Synthesis:GGR Nature Cell Biology8, 640 - 642 (2006)

  32. Repair Replication: M1 +/+ Nature Cell Biology8, 640 - 642 (2006)

  33. Repair Replication: M6 -/- Nature Cell Biology8, 640 - 642 (2006)

  34. Repair Replication Nature Cell Biology8, 640 - 642 (2006)

  35. Polymerase Activity-dependent Replication Repair Nature Cell Biology8, 640 - 642 (2006)

  36. NER is Reduced in Polk-/- Cells Nature Cell Biology8, 640 - 642 (2006)

  37. Removal of CPD and 6-4PP Nature Cell Biology8, 640 - 642 (2006)

  38. UV Removal Kinetics Nature Cell Biology8, 640 - 642 (2006)

  39. Proposed Model Nat Cell Biol. 2006 Jun;8(6):547-9

  40. Why is it Novel? • Y and B Family has two remarkably different properties • Processivity • Pol kappa ~ 1-5nts (?~25nt) • Pol epsilon ~ highly processive • Error-Rate • Pol Kappa: lacks exonuclease activity, 10-2 Error rate • Pol Epsion: < 10-5 error rate

  41. Do you think dNTPs concentration is a good trigger for Polymerase switching?

  42. Molecular Cell, Vol. 8, 213–224, July, 2001,

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