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
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
DNA Polymerase Families
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
DNA Polymerase Families
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
DNA Polymerase Families
DNA Polymerase Families
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Structures and Compositions
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Structures and Compositions
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Polymerization
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
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
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Polymerases are Highly Diverse
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Polymerases are Highly Diverse
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
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
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
DNA polymerases involved in DNA repair and replication
– Replicating Damaged DNA • Translesion Synthesis
– DNA Repair• Nucleotide Exision Repair
Functions of DNA polymerases
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Nat Cell Biol. 2006 Jun;8(6):547-9
Translesion Synthesis
Nat Cell Biol. 2006 Jun;8(6):547-9
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.
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)
Nature Cell Biology 8, 640 - 642 (2006)
RNA Synthesis Recovery:TCR
Nature Cell Biology 8, 640 - 642 (2006)
Unscheduled DNA Synthesis:GGR
Nature Cell Biology 8, 640 - 642 (2006)
Repair Replication: M1 +/+
Nature Cell Biology 8, 640 - 642 (2006)
Repair Replication: M6 -/-
Nature Cell Biology 8, 640 - 642 (2006)
Repair Replication
Nature Cell Biology 8, 640 - 642 (2006)
Polymerase Activity-dependent Replication Repair
Nature Cell Biology 8, 640 - 642 (2006)
NER is Reduced in Polk-/- Cells
Nature Cell Biology 8, 640 - 642 (2006)
Removal of CPD and 6-4PP
Nature Cell Biology 8, 640 - 642 (2006)
UV Removal Kinetics
Proposed Model
Nat Cell Biol. 2006 Jun;8(6):547-9
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,