Download - Molicular cell biology
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EUKARYOTIC DNA POLYMERASES
Course teacher :-
Dr. P.NagraJan Dr . R.Renuka
Professor
Presented by :-Kale Ravindra
Ramrao 09-607-05
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Eukaryotic Replication
Cell growth and division divided into phases: M, G1, S, and G2
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Flow of Genetic Information in the CellMechanisms by which information is transferred
in the cell is based on “Central Dogma”
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INTRODUCTION What is DNA polymerase ?
What is DNA replication ?
What is DNA proofreading ?
DNA polymerase families ? Family A e.g , mt DNA polymerase Family B e.g – DNA polymerase a,d,e Family C e.g – DNA pol III
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Family D
Family X e.g – pol β,pol µ,TdT Family Y e.g – translesion synthesis polymerase Family RT e.g - telomerase
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Eukaryotic DNA PolymeraseAt least 15 different polymerases are present in
eukaryotes (5 have been studied more extensively)
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The Eukaryotic Replication Fork
The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in Table 10.5.
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DNA polymerase function has the following requirements:
◦all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP
◦Mg2+
◦an RNA primer
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DNA Polymerase ReactionThe 3’-OH group at the end of the growing
DNA chain acts as a nucleophile.The phosphorus adjacent to the sugar is
attacked, and then added to the growing chain.
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EUKARYOTIC DNA POLYMERASE
Efficient machinery is required to maintain the genetic information.
DNA polymerases (pols) α, β, γ, δ, and ε are the key enzymes required to maintain the integrity of the genome.
DNA polymerases can be further subdivided into seven different families: A, B, C, D, X, Y, and RT.
The replicative pols α, δ and ε are related to pol II in E. coli and form the family B
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No homologues for E. coli pol III exist in eukaryotes
Pol β is a major base excision repair pol , in animals pol λ has an obvious role in meiosis-associated repair
pol µ is involved in somatic hyper mutation in lymph nodes
pol σ, that links DNA replication to the establishment of sister chromatid cohesion
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3D structure of the DNA-binding helix-turn-helix motifs in human DNA polymerase beta
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DNA polymerase alpha-primase
DNA polymerase activity without exonuclease proofreading
consist of four subunits (A, B, C, D)
expression of pol α (A subunit) takes place when inactive cell mitogenically activated to re-enter the cell cycle
pol α with strongly phosphorylated A and B subunits interacts with cyclin A and co-localizes in sites of ongoing DNA replication
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DNA polymerase deltaThe message level and enzyme
activity of pol δ are up-regulated when quiescent cells are induced to proliferate
transcription factors Sp1 and Sp3The characteristic feature of pol δ is
its tight coupling to the proliferating cell nuclear antigen (PCNA)
Pol δ is a major replicative polymerase
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minor role in base excision repair in yeast
gap-filling function in mammalian, long-patch base excision repair
a function for pol δ in recombination, double strand break repair, telomere maintenance and cell cycle checkpoint control
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DNA polymerase epsilonDNA polymerase epsilon (pol ε) was
first purified as DNA polymerase II in 1970
PCNA independent form of pol δ from calf thymus
Many of residues are important for nucleotide binding and/or template-primer stabilization
The mammalian pol ε has been purified as a dimeric enzyme
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Shows uniqe charcter in B classcatalytic properties and sensitivity to
inhibitors3’-5’ exonuclease activityPol ε does not need PCNA as an
auxiliary factor for processive DNA synthesis
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DNA polymerase switching and processing of an Okazaki fragment on the lagging strand
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Removal of dispalced okazaki initiator RNA by FEN1/RTH1 nuclease
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Mismatch repair
o Enzyme systems constantly moniter DNA looking for altered DNA
Example – UV radiation causes two adjacent
Thymines to form a Thymine dimer When found, nuclase enzymes
remove the TT dimer and a few surrounding nucleotides
DNA polymerase fills in the gap
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Proofreading and Repair◦ DNA replication takes place only once each
generation in each cell
◦ Errors in replication (mutations) occur spontaneously only once in every 109 to 1010 base pairs
◦ Can be lethal to organisms
◦ Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 104 to 105 base pairs
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DNA Polymerase Repair
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DNA Double strand break repair pathway
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CONCLUSION
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DISSCUSSION
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THANK YOU