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DNA Polymerization Has Three Stages. 1) Initiation 2) Priming 3) Processive Synthesis. Problems to overcome: DNA Polymerization. The two strands must be separated, and local DNA over-winding must be relaxed. The single stranded DNA must be prevented from

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Dna polymerization has three stages
DNA Polymerization Has Three Stages

1) Initiation

2) Priming

3) Processive Synthesis


Problems to overcome: DNA Polymerization

  • The two strands must be separated, and local DNA over-winding

  • must be relaxed. The single stranded DNA must be prevented from

  • re-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously in

the 5’ 3’ direction.

3’

3. A primer strand is required.

5’


Dna polymerization initiation
DNA Polymerization: Initiation

  • • DNA replication begins at a specific site.

  • •Example: oriC site from E. coli.

  • • 245 bp out of 4,000,000 bp

  • • contains a tandem array of three 13-mers; GATCTNTTNTTTT

  • Synthesis takes place in both directions from the origin (two replication forks)


E. coli replication origin

•GATC common motif in oriC

•AT bp are common to facilitate duplex unwinding


Dna polymerization initiation1
DNA Polymerization: Initiation

  • • DNA replication begins at a specific site.

  • •Example: oriC site from E. coli.

  • • 245 bp out of 4,000,000 bp

  • • contains atandem array of three 13-mers; GATCTNTTNTTTT

  • •GATC common motif in oriC

  • •AT bp are common to facilitate duplex unwinding

  • Synthesis takes place in both directions from the origin (two replication forks)


Enzymes involved in the initiation of DNA Polymerization

Enzyme Function

dnaA recognize replication origin and melts DNA duplexat several sites

Helicase (dnaB) unwinding of ds DNA

DNA gyrase generates (-) supercoiling

SSB stabilize unwound ssDNA

Primase (dnaG) an RNA polymerase, generates primers for DNA Pol



DNA helicase: proposed mechanism

A1

B1

Stryer Fig. 27.17


Problems to overcome: DNA Polymerization

  • The two strands must be separated, and local DNA over-winding

  • must be relaxed. The single stranded DNA must be prevented from

  • re-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously in

the 5’ 3’ direction.

3’

3. A primer strand is required.

(overall

direction)

5’


Lagging strand is synthesized in short fragments

(1000-2000 nucleotides long) using

multiple primers

3’

5’


Problems to overcome: DNA Polymerization

  • The two strands must be separated, and local DNA over-winding

  • must be relaxed. The single stranded DNA must be prevented from

  • re-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously in

the 5’ 3’ direction.

3’

3. A primer strand is required.

5’



What is the function of RNA priming?

  • DNA polymerase tests the correctness of the preceding base pair

  • before forming a new phosphodiester bond

  • de novo synthesis does not allow proofreading of the first

  • nucleotide

  • Low fidelity RNA primer is later replaced with DNA


Lagging strand synthesis in e coli
Lagging strand synthesis in E. coli

3'

5'

Okazaki fragment

5'

3'

Primase

3'

5'

3'

5'

5'

3'

DNA Pol III

RNA primer

3'

5'

5'

5'

3'

3'

DNA Pol I

Template DNA

3'

5'

3'

5'

New DNA

3'

5'

5'

3'

DNA Ligase

3'

5'

3'

5'


Dna synthesis
DNA Synthesis

Helicase

Gyrase

SSB

Primase

3'

5'

DNA

Pol

III

DNA

Pol

I

5'

DNA Ligase

5'

3'

3'

5'


E coli dna polymerase iii
E. coli DNA Polymerase III

  • Processive DNA Synthesis

  • The bulk of DNA synthesis in E. coli is carried out by the DNA polymerase III holoenzyme.

  • • Extremely high processivity: once it combines with the DNA and starts polymerization, it does not come off until finished.

  • • Tremendous catalytic potential: up to 2000 nucleotides/sec.

  • • Low error rate (high fidelity) 1 error per 10,000,000 nucleotides

  • Complex composition (10 types of subunits) and large size (900 kd)


Sliding clamp

clamp loader

Polymerase

Polymerase

  • 3'-5' exonuclease

E. coli Pol III: an asymmetrical dimer

Stryer Fig. 27.30


2 sliding clamp is important for processivity of pol iii
2 sliding clamp is important for processivity of Pol III


Lagging strand loops to enable the simultaneous replication

of both DNA strands by dimeric DNA Pol III

Stryer Fig. 27.33


Dna ligase seals the nicks
DNA Ligase seals the nicks

O

-O

P

O

DNA Ligase +

O-

(ATP or NAD+)

AMP + PPi

O

O

P

O

OH

O-

• Forms phosphodiester bonds between 3’ OH and 5’ phosphate

• Requires double-stranded DNA

• Activates 5’phosphate to nucleophilic attack by trans-esterification

with activated AMP


ENZYME

O

(+)H

N

2

Ade

P

O

O(-)

2.E-AMP + P-5’-DNA

O

AMP-O

5'-DNA

P

O

O

O

O-

5'-DNA

P

O

AMP-O

OH

OH

O-

DNA Ligase -mechanism

  • E + ATP  E-AMP + PPi

O

3.

DNA-3'

OH

DNA-3'

5'-DNA

+

O

P

O

O-

+

AMP-OH


Dna synthesis in bacteria take home message
DNA Synthesis in bacteria: Take Home Message

1) DNA synthesis is carried out by DNA polymerases with high fidelity.

2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’ 3’ direction.

3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand).

4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase


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