DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and...

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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.

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

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

DNA Polymerase Families

Structures and Compositions

Polymerization

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

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

Two-metal Mechanism

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

Polymerases are Highly Diverse

Functions of DNA Polymerases

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

DNA polymerases involved in DNA repair and replication

– Replicating Damaged DNA • Translesion Synthesis

– DNA Repair• Nucleotide Exision Repair

Functions of DNA polymerases

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

Translesion Synthesis

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

Y Family Structure

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

Nucleotide Excision Repair

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

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

Pol Kappa• Localization pattern

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

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

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,

Complementation of the NER deficiency by polκ

Nature Cell Biology 8, 640 - 642 (2006)

RNA Synthesis Recovery:TCR

Unscheduled DNA Synthesis:GGR

Repair Replication: M1 +/+

Repair Replication: M6 -/-

Repair Replication

Polymerase Activity-dependent Replication Repair

NER is Reduced in Polk-/- Cells

Removal of CPD and 6-4PP

UV Removal Kinetics

Proposed Model

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

Do you think dNTPs concentration is a good trigger for

Polymerase switching?

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

DNA Polymerases Nucleotide polymerizing enzymes that are central players in DNA repair and...

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