molicular cell biology

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 polymerase with proofreading ability

DNA Double strand break repair pathway

CONCLUSION

DISSCUSSION

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