DNA polymerases (DNA Pol)

Structural gene Polymeriza -tion rate (Nucleotide / sec) Processivity ( nucleotide added before polymerase dissociate Function Activity

Pol I

polA (dnaE)

16-20

3-200

DNA repair 5->3 polymerase, 3->5 exonuclease (proofreading) 5->3 exonuclease (RNA primer removal) damaged DNA repairing 3->5 exonuclease

Pol II Pol III

polB

40

1500

polC

250-1000

500,000

elongation 5 --> 3 DNA synthesis 3->5 exonuclease (proofreading)

Enzymes involved in Replication

Protein Pol I Prokaryotic / Eukaryotic
Prokaryotic

Activity
5->3 polymerase, 3->5 exonuclease (proofreading), 5->3 exonuclease (RNA primer removal)

Pol III
DNA Polymerase DNA Polymerase DNA Polymerase DNA Polymerase

Prokaryotic
Eukaryotic

5 --> 3 DNA synthesis 3->5 exonuclease (proofreading)

5->3 polymerase, complexes with primase then begins DNA Synthesis from RNA primers 5->3 polymerase, 3->5 exonuclease (proofreading), high processivity when complexed with PCNA. Mitochondrial DNA polymerase (5->3) 5->3 polymerase, high processivity, regulatory role

Eukaryotic

Eukaryotic Eukaryotic

Enzymes involved in Replication

Protein Primase Prokaryotic / Eukaryotic
Both

Activity
5->3 RNA polymerase, makes primers

DNA Helicase Single Stranded Binding Protein

Topoisomerase (Type I and II) DNA gyrase DNA ligase Initiator proteins Telomerase

Both
Both Both Prokaryotic Both Both Eukaryotic

Untwists DNA
Coats DNA to prevent strands re-anealing Relieve stress of supercoiling (Type I) and introduces negative supercoiling (Type II) (Type II topoisomerase Seals breaks in the DNA backbone between 3 OH and 5 PO4 Bind at the origin of replication Reverse transcriptase activity (5 to 3) using an endogenous RNA template

DNA replication: Prokaryotes Vs Eukaryotes

Differences in DNA replication in prokaryotes and eukaryotes exist due to differential genome organization

Prokaryotes
Circular genome Single piece genome

Eukaryotes
Linear genome Multipartite genome

Comparitively smaller genome

Larger genome: human

size: bacterial genome is 4.6X106 base pairs.

genome is 3X109 base pairs (600 times larger than E. coli genome).

DNA replication: Prokaryotes Vs Eukaryotes

Prokaryotes
One origin of replication

Eukaryotes
Many origins of DNA

replication
Much faster process (50,000 base

pairs / min).
Use Operon system.

Comparitively slower process

(2,000 base pairs / min).

Use the Tarnscription (from

nucleous)....>Translation (at the ribosome site in cytoplasm....> Protein synthesis (in cytoplasm).

Prokaryotes
The Pre-replication complex

Eukaryotes
The pre-RC is made up of the

(pre-RC) is made up of the following factors :

a) A helicase such as dnaA, which

following factors:

a) A six-subunit complex called

unwinds the DNA ahead of the replication fork.

b) Single stranded binding proteins

(SSBs) bind to each DNA strand to keep DNA unwound

c) A primase such as dnaG, which

generates an RNA primer to be used in DNA replication.

d) A DNA holoenzyme, which is

actually a complex of enzymes that performs the actual replication.

Origin Recognition Complex (ORC) which binds to the origin. b) Two regulatory proteins called Cdc6 and Cdt1 which are recruited by ORC. c) The MCMs (Minichromosome Maintenance proteins), the helicase complex. d) These proteins assemble on cellular origins in G1 phase of the cell cycle. Once these proteins are assembled, the MCMs are phosphorylated and DNA replication begins.

Prokaryotes

Eukaryotes
1.

Two DNA pol are involved in replication: 1. Pol I (DNA repair) 2. Pol III (DNA replication)

At least five DNA pol have been identified Mitochondrial DNA replication DNA Pol Nuclear DNA replication

2. DNA Pol (replication initiation) 3. DNA Pol , 4. DNA Pol (leading strand synthesis) 5. DNA Pol d (leading strand synthesis) Both Pol and Pol d have 3'->5' exonuclease (proofreading activity

Have less copies of replicating enzymes (E. coli has 10 to 20 molecules of DNA pol III) No end-replication problem due to circular DNA

More copies (20,000 and 60,000 molecules of pol ) Telomerase activity helps to solve endreplication problem

However, need for RNA primers , Synthesis of Okazaki fragments, removel and replacement of RNA primers and the ligation of adjacent daughter strand with DNA ligase are similar for both prokaryotes and eukaryotes