Chapter 2: DNA Synthesis (Replication)

Required reading: Stryer’s Biochemistry 5th edition p. 127-128, 750-754, 759-766, 768-773(or Stryer’s Biochemistry 4th edition p. 88-93, 799-809, 982-986, 809-814)

Normal recitation time: Thursdays 11-12, B-185 PWB

Exam: Wed., December 1 Proposed recitation time for this exam: Tue, Nov. 30, 11-12

DNA Polymerization Reaction

E. coli DNA Polymerases

Characteristic Pol I Pol II Pol III Mol. Weight (Da) 103,000 88,000 900,000

Number of polypeptides

1 4 10

Polymerase 5' 3' yes yes yes rate (nucleotides/sec) 16-20 7 250-1000 3' 5' exonuclease yes yes yes 5' 3' exonuclease yes no no

# molecules/cell 400 100 10 function Primer removal,

gap filling unknown Major replicative

polymerase

N C

E. coli DNA Polymerase I

Klenow Fragment

Polymerase3' 5‘ Nucl. 5' 3' Nucl.

36 kDa 67 kDa

• a large cleft for binding duplex DNA • flexible "finger" and "thumb" regions for positioning DNA duplex

and the incoming dNTP • polymerase site located in the "palm" region • 3' 5' and 5'- nuclease catalytic sites

Typical Polymerase Structure: E. Coli Pol I

thumb

palm

fingers

exonuclease

polymerase

Polymerase with bound DNA

Mechanism of phosphoryl transfer

Polymerase fidelity mechanisms

1. Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand.

2. H-bond formation between the minor groove of the new base pair and the amino acids in the polymerase active site.

3. Proofreading mechanism via 3' exonuclease that excises incorrectly added nucleotides.

1. Correct Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand induces conformational change required for polymerization reaction:

Thumb

Fingers

2. H-bond formation between the minor groove of the new base pair and amino acids in the polymerase active site:

All Watson-Crick base pairs contain two H-bond acceptors at the same sites of the

minor groove

HN

N

O

O

NN

N

NNH 2

A•TG•C

NH

N

NO

NH 2

NN

N

H2N

O

NH

N

N

O

NH2

N

NN

2NH2

O

C:G

HNN

O

O

N

N

N

NHN2

T:A

3. 3’-Exonuclease Proofreading function of DNA polymerases excises incorrectly added nucleotides.

Fidelity of DNA Polymerization: Absolutely Essential!!

Error Probability = Polymerization error (10-4)

X 3' 5' Nuclease error (10-3)

= 10-7 (1 in 10,000,000 nt)

DNA Polymerization Has Three Stages

1) Initiation

2) Priming

3) Processive Synthesis

Problems to overcome: DNA Polymerization

1. The two strands must be separated, and local DNA over-windingmust be relaxed. The single stranded DNA must be prevented fromre-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously inthe 5’ 3’ direction.

3’

5’

3. A primer strand is required.

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: 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)

Enzyme Function

dnaA recognize replication origin and melts DNA duplex at 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

Enzymes involved in the initiation of DNA Polymerization

Crystal structure of bacterial DNA helicase

Stryer Fig. 27.16

DNA helicase: proposed mechanism

Stryer Fig. 27.17

B1 A1

Problems to overcome: DNA Polymerization

1. The two strands must be separated, and local DNA over-windingmust be relaxed. The single stranded DNA must be prevented fromre-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously inthe 5’ 3’ direction.

3’

5’

3. A primer strand is required.

(overall direction)

Lagging strand is synthesized in short fragments (1000-2000 nucleotides long) using

multiple primers

3’

5’

Problems to overcome: DNA Polymerization

1. The two strands must be separated, and local DNA over-windingmust be relaxed. The single stranded DNA must be prevented fromre-annealing and protected from degradation by cellular nucleases.

2. Both antiparallel strands must be synthesized simultaneously inthe 5’ 3’ direction.

3’

5’

3. A primer strand is required.

A short stretch of RNA is used as a primer for DNA synthesis

(dnaG)

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

3' 5'

Primase

3'5'

5'

DNA Pol III

3'

5'5'

RNA primer

3'

3' 5'3'

DNA Ligase

5'

Template DNA

5' 3'Okazaki fragment

5' 3'

3' 5'3'5'

New DNA

3'5'

5'5' 3'

DNA Pol I

3'

3'

DNA Synthesis

3'

3'5'

SSB

5'

Leading Strand

5'

DNA Pol I

3'DNA Ligase

Lagging Strand

HelicaseGyrase

3'

5'

DNA Pol III

Primase

E. coli DNA Polymerase III

Processive DNA SynthesisThe 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)

E. coli Pol III: an asymmetrical dimer

Polymerase Polymerase

Stryer Fig. 27.30

clamp loader

Sliding clamp

3'-5' exonuclease

2 sliding clamp is important for processivity of Pol III

Stryer Fig. 27.33

Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III

DNA Ligase seals the nicks

OH P

O

-O O

O-

O P

O

O

O-DNA Ligase + (ATP or NAD+)

AMP + PPi

• 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

DNA Ligase -mechanism

1. E + ATP E-AMP + PPi

OH +DNA-3' P

O

AMP-O O

O-

5'-DNA O P

O

O

O-

DNA-3' 5'-DNA

+ AMP-OH

3.

2. E-AMP + P-5’-DNA P

O

AMP-O O

O-

5'-DNA

(+)H2NP

O

O(-)

O

OH

O

Ade

ENZYME

OH

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