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DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain.

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Presentation on theme: "DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain."— Presentation transcript:

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 Advances in Protein Chemistry, Vol. 69, , 2004 DNA Polymerase Families

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

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

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

6 Advances in Protein Chemistry, Vol. 69, , 2004

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

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

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

10 Science Dec 23;266(5193): Two-metal Mechanism

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 Dec 23;266(5193): Science Dec 23;266(5193):2022-5

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

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

14 Polymerases are Highly Diverse Advances in Protein Chemistry, Vol. 69, , 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 Advances in Protein Chemistry, Vol. 69, , 2004 DNA polymerases involved in DNA repair and replication

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

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

20 Translesion Synthesis Nat Cell Biol 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 Oct;12(5):529-36

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

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

24 Nucleotide Excision Repair Repair of Bulky Lesions (i)Recognition (ii)Separation of the double helix (iii) Incision (iv) Excision (v) Synthesis (vi) Ligation –Types Global Genome Repair Transcription Coupled Repair

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 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 |

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

29 Complementation of the NER deficiency by polκ Nature Cell Biology 8, (2006)

30 RNA Synthesis Recovery:TCR

31 Nature Cell Biology 8, (2006) Unscheduled DNA Synthesis:GGR

32 Nature Cell Biology 8, (2006) Repair Replication: M1 +/+

33 Nature Cell Biology 8, (2006) Repair Replication: M6 -/-

34 Nature Cell Biology 8, (2006) Repair Replication

35 Nature Cell Biology 8, (2006) Polymerase Activity-dependent Replication Repair

36 Nature Cell Biology 8, (2006) NER is Reduced in Polk-/- Cells

37 Nature Cell Biology 8, (2006) Removal of CPD and 6-4PP

38 Nature Cell Biology 8, (2006) UV Removal Kinetics

39 Proposed Model Nat Cell Biol 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, Error rate Pol Epsion: < 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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