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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 their integrity to ensure faithful transmission of genetic information from one generation to the next. biology of the cell / Bruce Alberts … [et al.].-- 4th ed.

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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Page 1: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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.

Page 2: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

DNA Polymerase Families

Page 3: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

DNA Polymerase Families

Page 4: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

DNA Polymerase Families

Page 5: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

DNA Polymerase Families

Page 6: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

DNA Polymerase Families

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 7: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Structures and Compositions

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 8: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Structures and Compositions

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 9: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Polymerization

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Molecular biology of the cell / Bruce Alberts … [et al.].-- 4th ed.

Page 10: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Science. 1994 Dec 23;266(5193):2022-5

Two-metal Mechanism

Page 11: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 12: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Polymerases are Highly Diverse

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 13: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Polymerases are Highly Diverse

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 14: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Polymerases are Highly Diverse

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 15: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Functions of DNA Polymerases

Page 16: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 17: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

DNA polymerases involved in DNA repair and replication

Page 18: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

– Replicating Damaged DNA • Translesion Synthesis

– DNA Repair• Nucleotide Exision Repair

Functions of DNA polymerases

Advances in Protein Chemistry, Vol. 69, 137-165, 2004

Page 19: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 20: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Translesion Synthesis

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

Page 21: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 22: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Y Family Structure

Mol Cell. 2001 Aug;8(2):417-26           

Page 23: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nucleotide Excision Repair

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

Page 24: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

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

Page 25: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 26: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Pol Kappa• Localization pattern

J Cell Sci. 2005 Jan 1;118(Pt 1):129-36

Page 27: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 29: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Complementation of the NER deficiency by polκ

Nature Cell Biology 8, 640 - 642 (2006)

Page 30: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

RNA Synthesis Recovery:TCR

Page 31: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Unscheduled DNA Synthesis:GGR

Page 32: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Repair Replication: M1 +/+

Page 33: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Repair Replication: M6 -/-

Page 34: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Repair Replication

Page 35: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Polymerase Activity-dependent Replication Repair

Page 36: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

NER is Reduced in Polk-/- Cells

Page 37: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

Removal of CPD and 6-4PP

Page 38: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Nature Cell Biology 8, 640 - 642 (2006)

UV Removal Kinetics

Page 39: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Proposed Model

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

Page 40: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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

Page 41: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

Do you think dNTPs concentration is a good trigger for

Polymerase switching?

Page 42: DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and replication, the processes that duplicate genomes and maintain

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