1.

Semiconservative

2. Conservative

3. Dispersive

DNA Replication
DNA DNA
DNA: Double helix and antiparallel 5 3
3 5
Semiconservative Meselsson & Stahl
Double helix DNA must be separated
Leading strand vs. Lagging strand
Okazaki fragments
In E.coli start point: OriC (Origin of replication)

DNA Replication
Required enzymes: - DNA polymerase I and III
- RNA primase (Primosome complex)
- DNA helicase
- DNA ligase
- DNA girase
Other proteins: - Single stranded binding protein
(SSBP)
- Initiator protein (dnaB)
Primer RNA

 Replication orientation: 5 3
Movement of Replication fork

Leading strand
5
(continous)
Lagging strand
(discontinous)

Okazaki fragment

Replication fork

Okazaki Fragments
In E. coli: 1000-2000 nt, in eukariot: 100-200 nt
Joined by DNA ligase

 Unwinding of DNA
DNA helicase

- catalyze unwinding process of helix DNA

- separate double stranded DNA
- 2 types in E.coli: Helicase II (lagging strand) &
Rep protein (leading strand)
- require ATP
5

Rep protein

Leading
strand

Helicase II

5
Lagging
strand

 Single-stranded DNA binding protein = SSBP

- Bind tightly to DNA

- Stabilize separation of double stranded DNA as
template
- no requirement of ATP

SSBP

 DNA girase (Topoisomerase II)

- Catalyze the synthesis of negative supercoil of DNA

- Essential for unwinding process
- Require ATP
Primer RNA
- Initiate the synthesis of DNA
- Synthesis of RNA primer is catalyzed by
primase & RNA polymerase

Primase
-6 other proteins Primosome
- BM 60 kD
- Initiate the synthesis of Okazaki fr. (lagging strand)
- Synergistic interaction with RNA polymerase
initiate the synthesis of leading strand

Primer RNA
3

3
5
3

- Length of primer RNA depends on species ca. 1-60 nt

E. coli: 10-60 nt
-After DNA synthesis began primer will be digested
DNA polymerase I
- Isolated in E.coli by Arthur Kornberg (1957)
- Single polypeptide, BM 103 kD
- Functions:
1. Polymerization (adding nt to 3-OH end of DNA)
(DNA) n + dNTP

(DNA) n+1 + PPi

Polymerization reaction
Required components:
- Precursor: dNTP (dATP, dGTP, dCTP, dTTP)
- Mg2+
- Primer RNA (ujung 3-OH bebas)
- template DNA
Orientation of polymerization reaction: 5 3

A
5

G
P

C
P

dGTP

PPi

OH

G
P

C
P

G
3

OH

DNA Polymerase I
*Addition of base complementary to template
* Synthesize only short DNA 20 nt
* Rate of synthesis: 10 nt/second

2. DNA Repair
* Exonuclease activity: 3 5
- Separate the false nucleotide in replication
- Proof read mechanism by DNA polymerase I
DNA replication very accurate

TA
TA

Hidrolisis oleh
eksonuklease 3 5
3

OH

TA
C

A
5

* Exonuclease activity 53
- Separate up to 10 nt from 5-end of single stranded
DNA
5
Hydrolysis by 53 exonuclease

A
C

(nick)

Hydrolysis by 53

TA

exonuclease

TA

TA

- Repair false nucleotide in double stranded DNA

- Role:
Repair of mutation caused by UV irradiation &
chemical mutagene
Digest primer RNA

DNA polymerase I
small fragment
N

exonuklease
53

large fragment (fr. Klenow)

exonuklease
35

polymerase

DNA polymerase III

- DNA replication
- BM 900 kD
-H oloenzyme, > 10 subunit protein
7 subunit core enzyme

- Functions:

Polimerization 5 3
* Subunit
* DNA synthesis up to thousands nt
* Synthesis rate: 1000 nt/second
Exonuclease 3 5
* Subunit
* Editor for DNA replication accuracy of replication
increase up to 200 x

D. DNA ligase
- Bind fr. Okazaki
- Catalyze the synthesis of phosphodiester bonds
between 3-OH end of one DNA and 5-P end of the
other DNA
- Require energy from hydrolysis:
NAD+ NMN+ + AMP (E. coli)
ATP PPi + AMP ( Eukaryot, bakteriophage T4)
- Reactions:
O

DNA ligase

atau DNA-3-O-P-O-5-DNA
DNA-3-OH + -O-P-O-5-DNA + ATP
+

O-

NAD

O-

Dimer komples leading & lagging strand are synthesized

simultaneously by a dimer komplex DNA pol. III enzyme
The lagging strand template is looped around, and each
replicative DNA polymerase moves 5 3 relative to its
strand, copying template and synthesizing a new DNA
strand

After Okazaki fragment has been completely

synthesized, the loop is loosened and the enzyme
will move to synthesize another fragment
Okazaki.

Termination
- In locus Ter (T)
- Ter locus contains of GTGTGTTGT sequences
bind to Tus protein termination of DNA synthesis
- Tus protein binds to Ter inhibition of DnaB helicase

OriC
Ter E
Ter D
Ter A

Ter F

Ter C
Ter B

1. Replication runs through Ter E, Ter D, Ter A

and stops at Ter C or Ter B or Ter F
2. Replication runs through Ter F, Ter B, Ter C
and stops at Ter A or Ter D or Ter E

Counterclockwise

rB
Te

Tus protein

Te

rD

Clockwise

Ter

r
Te

Tus protein