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.
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
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
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
Molecular biology of the cell / Bruce Alberts … [et al.].-- 4th ed.
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Two-metal Mechanism
Science. 1994 Dec 23;266(5193):2022-5
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
DNA polymerases involved in DNA
repair and replication
Advances in Protein Chemistry, Vol. 69, 137-165, 2004
Functions of DNA polymerases
– Replicating Damaged DNA
• Translesion Synthesis
– DNA Repair
• Nucleotide Exision Repair
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.
Nucleotide Excision Repair
•
Repair of Bulky Lesions
(i) Recognition
(ii) Separation of the double
helix
(iii) Incision
(iv) Excision
(v) Synthesis
(vi) Ligation
– Types
Nat Cell Biol. 2006 Jun;8(6):547-9.
•
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
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
Nature Cell Biology 8, 640 - 642 (2006)
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,