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
Eukaryotic Replication
Cell growth and division divided into phases: M, G1, S, and G2
Flow of Genetic Information in the CellMechanisms by which information is transferred
in the cell is based on “Central Dogma”
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
Family D
Family X e.g – pol β,pol µ,TdT Family Y e.g – translesion synthesis polymerase Family RT e.g - telomerase
Eukaryotic DNA PolymeraseAt least 15 different polymerases are present in
eukaryotes (5 have been studied more extensively)
The Eukaryotic Replication Fork
The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in Table 10.5.
DNA polymerase function has the following requirements:
◦all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP
◦Mg2+
◦an RNA primer
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.
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
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
3D structure of the DNA-binding helix-turn-helix motifs in human DNA polymerase beta
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
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
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
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
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
DNA polymerase switching and processing of an Okazaki fragment on the lagging strand
Removal of dispalced okazaki initiator RNA by FEN1/RTH1 nuclease
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
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
DNA Polymerase Repair
DNA Double strand break repair pathway
CONCLUSION
DISSCUSSION
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